Low viscosity highly concentrated suspensions

ABSTRACT

The present invent ion also provides a high concentration low viscosity suspension of an pharmaceutically acceptable solvent with one or more sub-micron or micron-sized non-crystalline particles comprising one or more proteins or peptides. Optionally one or more additives in the pharmaceutically acceptable solvent to form a high concentration low viscosity suspension with a concentration of at least 20 mg/ml and a solution viscosity of between 2 and 100 centipoise that is suspendable upon shaking or agitation, wherein upon delivery the one or more sub-micron or micron-sized peptides dissolves and do not form peptide aggregates syringeable through a 21 to 27-gauge needle.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of protein storageand delivery, and more particularly, to novel compositions and methodsof making highly concentrated protein suspension and precursors thereof.

BACKGROUND ART

Without limiting the scope of the invention, its background is describedin connection with the concentration of proteins. The use of proteinsand other polypeptides for therapeutics is on the rise in recent yearsas a way to expand and better treat patients since they are viewed to beless toxic and behave more predictably in vivo than other classes ofdrugs not naturally found in the body. Delivery of protein therapeuticshas been limited primarily to dilute large volume intravenous injectionsto deliver the high dose required (100-1000 mg) and to avoid physicaland chemical instabilities of proteins at high concentrations. Apotentially less invasive method of administration is subcutaneousinjection. Since the injection volume is limited to 1.5 ml, theconcentration of the protein therapeutic is often substantially above100 mg/ml. In addition to polypeptide stability, another major concernis the dramatic increase in viscosity for solution concentrationsgreater than 100 to 400 mg/ml due to protein interactions. If theprimary interactions are attractive protein-protein interactions due toelectrostatics, this increase in viscosity can been avoided by addingsodium chloride to increase the ionic strength of the solution and byvarying the buffer species and pH of the solution. At these highconcentrations, large excipient concentrations are often needed toprotect against denaturation. An alternative approach would be to form asuspension of an insoluble protein in a non-aqueous solvent. Theviscosity of highly concentrated suspensions can be much lower than forsolutions and require smaller excipient levels to stabilize the protein.However, for successful delivery with concentrated suspensions, theparticle size and suspension uniformity must be controlled in order toadminister an accurate and uniform dose.

To date, there are relatively few examples of suspensions of proteins innon-aqueous media for medicinal purposes. Highly viscous suspensions ofbovine somatotropin, marketed to increase milk production in dairy cows,and a bovine growth hormone releasing factor analog, used to releasesomatotropin from the cow's pituitary gland, are formulated in sesameoil and Miglyol oil, respectively. These viscous suspensions require alarge 14-16 gauge needle for injection, whereas the preferred needlesize for humans is between 25-gauge and 27-gauge. In addition, a fewnon-aqueous injections have been formulated as extended releaseformulations for the peptide insulin and very stable proteins such asprotein C and a proprietary monoclonal antibody with the aid ofviscosity enhancers and gel forming polymers in the presence of diluentssuch as benzyl benzoate or benzyl alcohol. However, these formulationsare syringeable only with a larger 21-gauge needle causing considerablepain upon injection leading to non-compliance and the high levels ofexcipients reduce the overall concentration of the protein in theformulation. Another option is to crystallize the protein or monoclonalantibody and form an aqueous suspension of the crystals. This approachhas been shown for three monoclonal antibodies and insulin. However,crystallization of high molecular weight proteins can be very difficultdue to the high degree of segmental flexibility, and is more feasiblefor small peptides that have a much lower degree of flexibility.

DISCLOSURE OF THE INVENTION

The present invention provides a method of making a high concentrationlow viscosity protein or peptide suspension by forming one or moresub-micron or micron-sized particles comprising one or more proteins orpeptides, adding optionally one or more additives to the one or moresub-micron or micron-sized particles and suspending the one or moresub-micron or micron micron-sized particles in a pharmaceuticallyacceptable solvent to form a high concentration low viscosity suspensionwith a concentration of at least 20 mg/ml and a solution viscosity ofbetween 2 and 100 centipoise that is suspendable upon shaking oragitation, wherein upon delivery the one or more sub-micron ormicron-sized peptides dissolves and do not form peptide aggregates oronly a small fraction of aggregates and is syringeable through a 21 to27-gauge needle. The pharmaceutically acceptable solvent may be apharmaceutically acceptable aqueous solvent, a pharmaceuticallyacceptable non-aqueous solvent or combination.

In addition, the one or more micron-sized peptide particles are formedin a dosage container and may be delivered directly from the dosagecontainer that is a vial, an ampule, a syringe or a bulk container. Theone or more micron-sized peptide particles may be made by milling,precipitation, dialysis, sieving, spray drying, lyophilization, sprayfreeze drying, spray freezing into liquids, thin film freezing, orfreezing directly in a dosage container. The one or more additives maybe part of the one or more sub-micron or micron-sized particles, the ahigh concentration low viscosity suspension or both.

The present invention also provides a high concentration low viscositysuspension of an pharmaceutically acceptable solvent with one or moresub-micron or micron-sized non-crystalline particles comprising one ormore proteins or peptides. Optionally one or more additives in thepharmaceutically acceptable solvent to form a high concentration lowviscosity suspension with a concentration of at least 20 mg/ml and asolution viscosity of between 2 and 100 centipoise that is suspendableupon shaking or agitation, wherein upon delivery the one or moresub-micron or micron-sized peptides dissolves and do not form peptideaggregates or only a small fraction of aggregates syringeable through a21 to 27-gauge needle. The pharmaceutically acceptable solvent may be apharmaceutically acceptable aqueous solvent, a pharmaceuticallyacceptable non-aqueous solvent or combination. In addition, the one ormore micron-sized peptide particles are formed in a dosage container andmay be delivered directly from the dosage container that is a vial, anampule, a syringe or a bulk container. The one or more micron-sizedpeptide particles may be made by milling, precipitation, dialysis,sieving, spray drying, lyophilization, spray freeze drying, sprayfreezing into liquids, thin film freezing, or freezing directly in adosage container. The one or more additives may be part of the one ormore sub-micron or micron-sized particles, the a high concentration lowviscosity suspension or both.

The present invention provides a single dose high concentration lowviscosity suspension in a single dose container. The single dosecontainer includes a pharmaceutically acceptable solvent disposed in thesingle dose container, wherein the pharmaceutically acceptable solventis selected from an aqueous solvent, a non-aqueous solvent orcombination thereof and one or more sub-micron or micron-sizednon-crystalline particles disposed in the single dose container, whereinthe one or more sub-micron or micron-sized non-crystalline particlescomprising one or more proteins or peptides. In addition, one or moreadditives may be optionally disposed in the single dose container toform a high concentration low viscosity suspension with a aconcentration of at least 20 mg/ml and a solution viscosity of between 2and 100 centipoise syringeable through a 21 to 27-gauge needle.

DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A-C are SEM images of particles made by the process of oneembodiment of the present invention.

FIGS. 2A-2E are images of suspensions before (FIGS. 2A and B) and after(FIGS. 2C-E) centrifugation (20 min at 3000 rpm) from left to right 1.5MAmmonium Sulfate (a and c), 30% PEG300 (b and d), 35% NMP (e) all in 150mM pH 4.7 acetate buffer with added NaCl to 154 mM ionic strength.

FIG. 3 is an image of aqueous PEG 300 suspensions after 20 min ofcentrifugation at 3000 g. From left to right 30% PEG300, 40% PEG300, and50% PEG 300 all in 150 mM pH 4.7 acetate buffer with added NaCl to 154mM ionic strength.

FIG. 4 is a graph of the apparent viscosity of aqueous suspensions atvarious pHs all with 50% PEG 300 and added NaCl to 154 mM ionicstrength.

FIG. 5 is an image of aqueous 50% PEG 300 suspensions after 20 min. ofcentrifugation at 3000 g. From left to right pH 4.7 acetate buffer, pH5.5 acetate buffer, and pH 7.4 acetate buffer.

FIG. 6 is a graph of the apparent viscosity of aqueous suspensions withPEG 300 and organic additives in 150 mM pH 4.7 acetate buffer with addedNaCl to 154 mM ionic strength.

FIGS. 7A and 7B are graphs that show the apparent viscosities of milledBSA and trehalose at various ratios in 150 mM pH 4.7 acetate bufferalong with the theoretical viscosity as calculated from theKrieger-Dougherty equation using the [η] of the pure milled particles ina) 25% PEG 300 20% Ethanol and b) 35% PEG 300 and 15% NMP.

FIGS. 8A-8E are SEM images of various frozen powders of IgG.

FIG. 9 is a graph of the optical density of the IgG with variousadditives totaling 50% of the solvent by volume to decrease thesolubility of the IgG as measured at 350 nm. The right hand side has theabsorbance of the 5 mg/ml concentration of IgG in the pH 6.4 20 mMhistidine buffer with no additional additive.

FIG. 10A-10D are images of various suspensions of IgG.

FIG. 11A-11E are microscope images of various suspensions of IgG.

FIG. 12. Volume % of particles versus size measured for the originalmilled particles in acetonitrile and ethanol and after 2 months ofstorage for the suspensions in pure benzyl benzoate and a mixture ofbenzyl benzoate and oil both measured immediately after being diluted inethanol to 10-15% obscuration by light scattering.

FIG. 13A-13C Pictures of the 300 mg/mL Lysozyme suspension in 50/50Benzyl Benzoate and Safflower Oil.

FIG. 14 Viscosity of a solution of benzyl benzoate and safflower oil atroom temperature at varying concentrations.

FIG. 15 The apparent viscosity as a function of concentration ofparticle as suspensions in the non-aqueous solvents and the theoreticalviscosity of an aqueous lysozyme solution.

FIG. 16 Karl Fisher moisture content analysis of the suspensions.

FIG. 17 Image of a suspension taken immediately after formulation.

FIG. 18 Freezing temperature profiles of lysozyme solutions (10 mg/ml)inside vials.

FIG. 19A-19D Volume size distributions of the protein particles producedby Film Freezing in Vials in the conditions described in Table 2.

FIG. 20 shows a 4 ml of lysozyme solution (20 mg/ml) in water werefrozen by film freezing inside the vial.

FIG. 21 shows a 2 ml of hemoglobin solution (150 mg/ml) in water wasfrozen by film freezing inside the vial.

DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein, the term “high protein concentration” refers to liquids,gels, hydrogels or gel-like compositions with a protein concentration ofgreater than 100 mg/ml.

As used herein, the term “non-aggregating” or “not aggregating” or “notaggregated” refers to protein particles that remain in suspensiondespite being provided in the form of a high protein concentration,e.g., a protein concentration greater than 100 mg/ml.

As used herein, the term “syringable” refers to a final composition fordelivery to a subject that is sufficiently fluid to be flowable. Forexample, a composition that is “syringable” has a low enough viscosityto load the syringe and inject a subject from the syringe without undueforce.

As used herein, the term “non-settling” or “redispersible” refers to acomposition that remains in solution phase (i.e., they do not sediment)after an extended period of time, e.g., 1 hour, 2 hours, 1 day, 3 days,5 days, 1 week, 1 month, 3 months, 6 months, 1 year or more. Forexample, a composition is “re-dispersible” is upon re-dispersion it doesnot flocculate so quickly as to prevent reproducible dosing of a drug.

As used herein, the terms “protein(s),” “polypeptide(s)” and“peptide(s)” refers to a polymer composition formed from the linkingamino acids into a chain of various lengths.

As used herein, the term “additive(s)” refers to salts, sugars,organics, buffers, polymers and other compositions that include:Disodium edetate, Sodium chloride, Sodium citrate, Sodium succinate,Sodium hydroxide, Sodium glucoheptonate, Sodium acetyltryptophanate,Sodium bicarbonate, Sodium caprylate, Sodium pertechnetate, sodiumacetate, sodium dodecyl sulfate, aluminum hydroxide, aluminum phosphate,ammonium citrate, calcium chloride, calcium, potassium chloride,potassium sodium tartarate, zinc oxide, zinc, stannous chloride,magnesium sulfate, magnesium stearate, titanium dioxide,DL-lactic/glycolic acids, asparagine, L-arginine, argininehydrochloride, adenine, histidine, glycine, glutamine, glutathione,imidazole, protamine, protamine sulfate, phosphoric acid, Tri-n-butylphosphate, ascorbic acid, cysteine hydrochloride, hydrochloric acid,hydrogen citrate, trisodium citrate, guanidine hydrochloride, mannitol,lactose, sucrose, agarose, sorbitol, maltose, trehalose, surfactants,polysorbate 80, polysorbate 20, poloxamer 188, sorbitan monooleate,triton n101, m-cresol, benyl alcohol, ethanolamine, glycerin,phosphorylethanolamine, tromethamine, 2-phenyloxyethanol, chlorobutanol,dimethylsulfoxide, N-methyl-2-pyrrolidone, propyleneglycol, Polyoxyl 35castor oil, methyl hydroxybenzoate, tromethamine, cornoil-mono-di-triglycerides, poloxyl 40 hydrogenated castor oil,tocopherol, n-acetyltryptophan, octa-fluoropropane, castor oil,polyoxyethylated oleic glycerides, polyoxytethylated castor oil, phenol(antiseptic), glyclyglycine, thimerosal (antiseptic, antifungal),Parabens (preservative), Gelatin, Formaldehyde, Dulbccco's modifiedeagles medium, Hydrocortisone, Neomycin, Von Willebrand factor,Gluteraldehyde, Benzethonium chloride, White petroleum,p-aminopheyl-p-anisate, monosodium glutamate, beta-propiolactone,Acetate, Citrate, Glutamate, Glycinate, Histidine, Lactate, Maleate,Phosphate, Succinate, Tartrate, Tris, Carbomer 1342 (copolymer ofacrylic acid and a long chain alkyl methacrylate cross-linked with allylethers of pentaerythritol), Glucose star polymer, Silicone polymer,Polydimethylsiloxane, Polyethylene glycol, carboxymethylcellulose,Poly(glycolic acid), Poly(lactic-co-glycolic acid), Polylactic acid,Dextran 40, Poloxamers (triblock copolymers of ethylene oxide andpropylene oxide),

For highly concentrated protein suspensions in non-aqueous solvents, theKrieger-Dougherty equation can be used to correlate the relativeviscosity of a suspension η over that of a solution η_(o) to the volumefraction of particles φ (Eq. 1).

$\begin{matrix}{\frac{\eta}{\eta_{o}} = \left\lbrack {1 - \left( \frac{\varphi}{\varphi_{\max}} \right)} \right\rbrack^{{- {\lbrack\eta\rbrack}}\varphi_{\max}}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

The intrinsic viscosity, [η] approaches 2.5, the Einstein value,assuming non-interacting, spherical particles with only excluded volumeinteractions. However, [η] increases upon solvation of the particles,deviation from a spherical shape and electrostatic interactions thatproduce primary, secondary and tertiary electroviscous effects. For thenon-aqueous protein suspensions demonstrated previously, [η] ofapproximately 2.5 indicated a lack of solvation, shape andelectroviscous effects on the viscosity of lysozyme milled particles.However, non-aqueous solvents can sometimes cause pain on injection anddelayed and slowed release of the particles. Consequently, aqueous-basedsuspensions of highly concentrated and molecularly stable, proteinparticles would be an attractive alternative to non-aqueous suspensions.

Therapeutic proteins may be designed for high solubilities in aqueousmedia in the range of 100 mg/ml, for example, for the model protein BSA.Thus, the solubilities must often be decreased significantly, in orderto form a suspension of micron-sized particles. Precipitant that candecrease the solubility of a protein in water may be separated intothree categories; salts, polymers and water-soluble organics. Salts maydecrease the solubility of a protein by competing for waters ofhydration as well as ion binding to produce stronger interactionsbetween protein molecules. Salts also reduce electrostatic repulsion bydecreasing the thickness of the double layer. However, a total ionicstrength of around 154 mM, the tonicity of the blood, is typicallyrecommended to prevent pain upon injection. Polymers, most commonlypolyethylene glycol (PEG), which are preferentially excluded from theprotein surface, produce depletion attraction causing precipitation. PEGis also known to increase the thermal stability of a protein.Furthermore, it is an acceptable excipient for sub-cutaneous injection.Water-soluble organic additives, such as ethanol andn-methyl-2-pyrrolidone (NMP), decrease protein solubility by loweringthe dielectric constant and by excluded volume effects resulting fromtheir exclusion from the protein surfacc.

The objective of this study was to form low viscosity (<50 cP), highlyconcentrated (100 to 350 mg/ml) aqueous suspensions of sub-micron tomicron-sized particles of the model protein bovine serum albumin (BSA)suitable for subcutaneous delivery. Precipitants, known to reduce thesolubility of proteins included combinations of ammonium sulfate (arepresentative salt), PEG300 (a low molecular weight polymer), andethanol and N-methyl-2-pyrrolidone (NMP). Two key factors influence theviscosity of the suspension at a given volume fraction of protein: theinitial viscosity of the solution without protein and the intrinsicviscosity with protein present. Milled particles smaller than 37 μm ofBSA were suspended in a variety of aqueous-based solvents tocharacterize these competing effects. In many cases, the concentrationsof additives were within pharmaceutically acceptable limits. Theapparent viscosity of the suspension at various particle concentrationsis correlated with the Krieger-Dougherty equation to determine theintrinsic viscosity. The intrinsic viscosity was used to characterizeinterparticle interactions including electroviscous and solvationinteractions. A variety of low viscosity, highly concentrated (up to 350mg/ml) aqueous-based suspensions comprising pharmaceutically relevantadditives are reported for milled BSA particles. The insight gained fromthe study of the viscosities and morphologies of suspensions of themodel protein BSA will be useful for the design of suspensions oftherapeutic proteins such as IgG.

BSA powder or trehalose powder as received was dry milled with aporcelain mortar and pestle separately for several minutes. The milledpowder was then sieved through a number 400 mesh and particles smallerthan 37 μm were collected. For the mixed milled BSA and trehaloseparticles, the necessary ratio of BSA to trehalose was then mixed withthe mortar and pestle and received through the number 400 mesh topreserve the correct particle ratio but retain the smaller than 37 μmparticle size. Known weights of powder were suspended in aqueoussolutions composed of NMP, PEG 300, and/or ethanol, and sodium chlorideor ammonium sulfate salts. The pH was chosen as 4.7, 5.5 or 7.4 with anacetate (pH 4.7 and 5.5) or phosphate (pH 7.4) buffer. Each vial wasthen shaken by hand to disperse the powder evenly through thesuspension. Additional mixing using the tip of the needle was used toensure uniformity if necessary.

BSA was readily soluble at 5 mg/ml in pH 4.7 150 mM acetate buffer. Analiquot of a 5 mg/ml BSA solution was mixed with an equal volume of asecond aqueous solution containing additives for the purpose ofdetermining the degree to which the protein precipitated (either PEG300,NMP or a combination of the two). The precipitation of the protein wasclassified as highly turbid (HT), lowly turbid (LT, slight turbidity),or no change in turbidity (N). The solutions were all formulated in a pH4.7 150 mM acetate buffer.

The apparent viscosity of the IgG suspensions was measured as the timeto draw 0.25 mL of the suspension into a 25 gauge 1.5″ needle attachedto a 1 ml tuberculin slip tip syringe. Typical times ranged from 5 to100 s. Each measurement was made at least 3 times and averaged, whilemaintaining the suction force by holding the end of the plunger at the 1ml mark each time. A linear correlation curve for viscosity and time todraw 1 mL (4 times the amount measured) was constructed from measuringliquids of known viscosity (PEG200, PEG 300, PEG 400, water, ethanol,olive oil, and benzyl benzoate). This correlation, as expected from theHagen-Poiseuille equation, gives an r² value greater than 0.999 and wasreported previously. In most cases the reproducibility in viscosity waswithin 5%. In our experiments a maximum volume of 25% of the cavity inthe syringe was filled with suspension for the uptake. Consequently, thepressure drop was relatively constant. The error introduced by the smallchange in pressure drop was minimized by using the same plunger positioneach time and by correlating the data to liquids of known viscosity.

Following the suspension viscosity measurement, the protein suspensionswere centrifuged for 20 min at 3000 rpm using a rotating bucketcentrifuge rotor (part A-4-62) with a 2 ml centrifuge tube adapter foran Eppendorf Centrifuge (model 5810, Wesbury, N.Y.). The centrifugedsamples were photographed and the supernatant was separated by carefullydecanting the sample using a needle and syringe. The remaining proteinparticles were then redispersed in ˜15 ml of acetonitrile under gentlebath sonication for 5 min. The dissolution of BSA in acetonitrile wasnegligible. After redispersion, the protein particle size was thenmeasured for a drop of the sonicated dispersion. The dispersion wasdiluted to an obscuration of approximately 10% in acetonitrile in asmall-volume (11 ml) magnetically stirred cell and the particle size wasanalyzed by light scattering using the Malvern Instruments MastersizerS.

After the centrifugation described in the Particle Size Measurementsection, the recovered supernatant was then filtered through a 0.22 μmfilter and collected. The filtered sample was then diluted to a totalvolume of 0.7 ml in pH 4.7 150 mM acetate buffer. Three 200 μl aliquotsof each sample were then placed on a UV-transparent 96-well plate andimaged at 280 nm using a spectrophotometer. A standard curve of BSAconcentrations of 3, 2, 1, 0.5, and 0 mg/ml in the same buffer versusabsorbance at 280 nm yielded an r² value greater than 0.99. Thecalibration curve was used to regress the soluble concentration valuesfor each sample. If necessary, the sample was subsequently diluted andremeasured till the concentration fell in the range of 0.5-3 mg/ml.

A drop of the aqueous suspension was flash frozen onto frozen aluminumSEM stages maintained at −200° C. with liquid nitrogen. The frozendroplet was lyophilized with 12 hours of primary drying at −40° C. thatwas followed by a 6 hour ramp to 25° C. and secondary drying for atleast 6 hours at 25° C. using a VirTis Advantage Plus XL-70 shelflyophilizer. The lyophilization produced a dried powder sample on theSEM stage. Dry powder samples of the milled particles were placed onadhesive carbon tape. Each sample was then gold-palladium sputter coatedusing a Cressington 208 bench top sputter coater to a thickness of 15nm. Micrographs were then taken using a Zeiss Supra 40 VP scanningelectron microscope with an accelerating voltage of 5 kV.

Various additives can be added to decrease the solubility of a proteinin aqueous buffer. The turbidimetric studies of solubility in Table 1indicate that BSA precipitates with either pure PEG and NMP or mixturesthereof even at low protein concentration of 5 mg/ml. Table 2 indicatesthat PEG is a stronger antisolvent than NMP as a low turbiditysuspension (LT) is formed at 30%. Furthermore, mixtures of the twoantisolvents can produce synergistic effects on precipitating protein.For example, a 20-20% mixture causes high turbidity whereas 40% NMP doesnot produce a change in turbidity. Finally for a given total weight % ofantisolvent, the turbidity increases as the relative fraction of PEGincreases. These experiments indicate that even a dilute 5 mg/ml proteinconcentration is well above the solubility limit with theseantisolvents. Therefore, only a small fraction of the protein will bedissolved with these additives when the overall protein concentration ison the order of 200 mg/ml, a typical concentration for the injectablesuspensions.

FIGS. 1A-C are SEM images of particles made by the process of oneembodiment of the present invention. FIG. 1A is a SEM image of originalmilled particles. FIG. 1B is a SEM image of 30% PEG300 suspension at 200mg/ml flash frozen and lyophilized. FIG. 1C is a SEM image of 25% PEG30020% ethanol suspension at 350 mg/ml flash frozen and lyophilized.

The morphology of the original milled particles is shown in FIG. 1A. Theaverage particle size was 20 μm. The size was chosen to be small enoughto pass through a 25-27 gauge needle with an inside diameter of smallerthan 241 μm. A drop of the final suspension was frozen and lyophilized,and SEM was utilized to determine the particle size in the suspensionsfor two choices of precipitants shown in FIGS. 1B and C. The particlesizes were only modestly larger than that of the original milledparticles indicating little growth from particle aggregation or Ostwaldripening. In addition, FIG. 1C shows a slight increase in the amount ofsmaller sub-1 μm particles. These results indicate that the particlesize could essentially be maintained in the suspensions, and thatdissolution of the protein was minimal.

The purpose of this section is to describe the mechanisms for proteinprecipitation and to provide a brief overview of the results for theviscosities for each class of precipitant. To form a suspension of BSAin aqueous media, the media must be designed to prevent a significantfraction of the BSA from dissolving. The solubility of BSA in a pureaqueous buffer at pH of 4.5 is greater than 100 mg/ml. Various additiveswere introduced to the aqueous buffer media to decrease the solubilityincluding salts, polymers, and water soluble organics.

Table 1 is a table of the precipitation of BSA in PEG and NMP mixturesat 5 mg/ml. (N indicates transparent solution with no change inturbidity versus case without additives, LT indicates low turbidity, HTindicates a high level of turbidity)

PEG % NMP % 0 5 10 15 20 25 30 35 40 45 50 0 N N N N N N LT LT HT HT HT5 N N N N N N LT LT HT HT 10 N N N N N LT HT HT HT 15 N N N N LT HT HTHT 20 N N N N HT HT HT 25 N N N LT HT HT 30 N N N HT HT 35 N N HT HT 40N LT HT 45 N HT 50 N

The mechanisms by which the solubility is lowered is given for each typeof precipitant in Table 2. Formation of an opaque white concentratedsuspension at 200 mg/ml was possible for all three various additivegroups as seen in Table 1. Table 2 is a comparison of differentadditives to lower solubility of a protein, suggested mechanism for thedecrease in solubility and the suspension viscosity measured at 200mg/ml in 150 mM pH 4.7 acetate buffer plus each additive.

Example of Suspension Viscosity for 200 mg/ml Additive Mechanism tolower protein solubility milled BSA Salts Competition for waters ofhydration 3 cP for 1.5M Ion binding changing protein-protein (NH₄)₂SO₄interaction Decrease in electrostatic repulsion with a decrease in thedouble layer thickness Polymers Preferentially Preferential exclusion 18cP 30% from protein surface leading to depletion PEG300 attraction Lowersolvent dielectric constant Water Soluble Lower solvent dielectricconstant 10 cP for Organics Exclusion of solvent from protein 35% n-surface produces excluded volume methyl-2- effects pyrrolidone (NMP)

A summary of select viscosity results measured with a 25 g 1.5″ needle,which are described in greater detail below, is also presented in Table2. An extremely low viscosity of 3 cp was obtained with 1.5M (NH₄)₂SO₄.The values were also quite low for the other PEG and NMP antisolvents.All three of these examples are well within the limits of what would beconsidered easily syringeable, since it would take less than 20 secondsto expel 1 ml from a 26 g needle. These results will be examined in muchgreater detail below in context of the morphologies of the particles andof the suspensions and for a much wider range of conditions.

FIGS. 2A-2E are images of suspensions before (FIGS. 2A and B) and after(FIGS. 2C-E) centrifugation (20 min at 3000 rpm) from left to right 1.5MAmmonium Sulfate (a and c), 30% PEG300 (b and d), 35% NMP (e) all in 150mM pH 4.7 acetate buffer with added NaCl to 154 mM ionic strength. Asshown in FIG. 2, the various solubility-decreasing additives produceddifferent amounts of suspended large particles. The relative quantity ofsuspended particles is evident qualitatively from the turbidity of theinitial suspension. An opaque white suspension was formed for 30% PEG300and 35% NMP as shown in FIGS. 2B and 2E suggesting a large amount ofsuspended particles relative to dissolved protein. This observation isconfirmed more quantitatively by the volume fraction of the precipitantafter 20 minutes of centrifugation at 3000 rpm, indicating very littleof the protein was soluble. This force is sufficient to settle out allparticles greater than ˜400 nm. These results support the directobservation of micron-sized protein particles in the suspensions bycryo-SEM in FIG. 1C. For 1.5M ammonium sulfate salt as an additive, theoriginal suspension was only translucent indicating a much smallerdegree of precipitation. After centrifugation, only a small volume ofprecipitate was present (FIGS. 2A and C) consistent with the observationof relatively low turbidity in the original suspension. The degree ofprecipitation as characterized by the turbidity of the initialsuspension and the volume fraction of precipitant after centrifugationwill be an important factor for understanding the viscosity behavior ofthe suspensions.

As shown in Table 2, an aqueous suspension with 30% PEG300 gives anapparent viscosity of 18 cP for 200 mg/ml BSA. This low viscosity may beexamined in terms of the presence of protein particles relative todissolved protein in solution. As has been demonstrated previously, wththe addition of 30% PEG300, the solubility of BSA is reduced fromapproximate 6 mg/ml at 25%, to 1 mg/ml. This large decrease insolubility was verified in Table 1 by the increase in turbidity for alevel of precipitant between 25% and 30% (by volume) PEG300 at a proteinconcentration of 5 mg/ml.

Suspensions of the pure milled BSA particles were formed for PEG300levels between 30 and 50% by volume. Two key factors influence theviscosity of the suspension at a given volume fraction of protein: theinitial viscosity of the solution without protein and the intrinsicviscosity with protein present. Table 3 shows the Intrinsic Viscositydata of suspensions to compare effects of electroviscous and hydration,all samples at pH 4.7 acetate 150 mM ionic strength buffer (unlessindicated otherwise). For intrinsic viscosity measurement, φ_(max)assumed to be 0.64. (NMP—N-methyl-2-pyrrolidone, ND—not determined) Theaverage particle diameter was determined by static light scattering was20 μm.

Suspension Apparent Solution Viscosity at 250 mg/ml Intrinsic AverageSoluble Viscosity BSA Viscosity Particle Concentration Solvent system(cP) (cP) [η] Diameter (μm) (mg/ml) 30% PEG300 2.6 56 11.4 9.6 ND 40%PEG300 4.6 85 9.6 17.4 2.0 50% PEG300 6.6 49 7.9 18.5 2.1 pH 5.5 50%PEG300 6.2 47 8.4 18.4 ND pH 7.4 50% PEG300 6.0 57 9.0 ND ND 40% PEG300and 10% 5.1 22 6.0 20.2 2.9 NMP 35% PEG300 and 15% 4.8 20 5.9 19.1 2.9NMP 25% PEG300 and 20% 3.6 18 6.5 19.5 2.3 NMP 25% PEG300 and 20% 3.3 145.8 7.4 4.8 Ethanol

As shown in Table 3, the solution viscosity (without protein) increasesfrom 2.6 for a 30% PEG300 solution to 6.6 for a 50% PEG300 solution. Atthe same time however, the intrinsic viscosity decreased from 11.4 to7.9. Consequently the overall suspension viscosity went down about 10%,a value twice that of the experimental uncertainty. Interestingly, theviscosity was higher at 40% PEG than 30%, as the intrinsic viscosity didnot decrease enough to compensate for the higher solvent viscosity(without protein). Thus, the amount of precipitant may be optimized tobalance its effect on the initial viscosity of the solution withoutprotein versus the intrinsic viscosity of the protein suspension.

FIG. 3 is an image of aqueous PEG 300 suspensions after 20 min ofcentrifugation at 3000 g. From left to right 30% PEG300, 40% PEG300, and50% PEG 300 all in 150 mM pH 4.7 acetate buffer with added NaCl to 154mM ionic strength. All 3 of the PEG-based suspensions after 20 min ofcentrifugation at 3000 g exhibited a high degree of large settledparticles (FIG. 3). However, for the 30% PEG300 suspension, thesupernatant appears slightly turbid, indicating the presence of smallsuspended nanoparticles. Thus, a soluble concentration was notdetermined for this case. The soluble concentration for the 40 and 50%PEG300 suspensions was between 2.0-2.1 mg/ml indicating that 99% of theparticles at 200 mg/ml were suspended (Table 3). The average particlediameters for the 40 and 50% PEG300 suspensions were both near theoriginal 20 μm value for the milled particles, as shown in table 3.However, for the 30% PEG300 suspension, the average particle diameterdecreased to ˜10 μm consistent with greater dissolution on the basis ofthe turbidity of the supernatant. This decrease in size is consistentwith the observation by SEM in FIG. 1. These smaller particles withhigher surface area, and dissolved protein contributed to the higherintrinsic viscosity in Table 3.

For the 30% PEG300 formulation, additional apparent suspensionviscosities were measured at higher ionic strengths, shown in Table 4.As the ionic strength of the solution increased, the viscosity decreasedfrom 56 to 19. The decrease in the electroviscous effects with anincrease in ionic strength and a decrease in the Debye lengthcontributes significantly to the decrease in intrinsic viscosity andthus the suspension viscosity.

Ionic strength (mM) Apparent Viscosity (cP) 150 56 300 23 500 19

To change the charge of the protein and the solubility, the solution pHwas increased from the pI of BSA (4.7) to a pH of 5.5, while maintainingthe acetate ion as the buffer. In addition, a phosphate buffer ion wasused as opposed to acetate buffer ion to raise the pH of 7.4. Since thesolubility of a protein increases as the pH moves away from the pI, the50% PEG300 additive was included in the media to ensure a low solubilityof BSA.

FIG. 4 is a graph of the apparent viscosity of aqueous suspensions atvarious pHs all with 50% PEG 300 and added NaCl to 154 mM ionicstrength. As shown in FIG. 4, neither the buffer ion nor the pH made asignificant change in the apparent suspension viscosity at anyconcentration. The solution viscosities for the varying pHs with 50%PEG300 varied only slightly from 6.0-6.6. Since the apparent viscositiesfor all cases at all concentrations were similar, the calculatedintrinsic viscosities increased only slightly from 7.9 to 9.0 for theincrease in pH. The theoretical curves in FIG. 4 were determined byregressing the intrinsic viscosity (Table 3) with the Krieger-Doughertyequation.

FIG. 5 is an image of aqueous 50% PEG 300 suspensions after 20 min. ofcentrifugation at 3000 g. From left to right pH 4.7 acetate buffer, pH5.5 acetate buffer, and pH 7.4 acetate buffer. As shown in FIG. 5, thesupernatant is still turbid for both the pH 5.5 and pH 7.4 samples aftercentrifugation. As a result, the soluble concentration could not bedetermined and the average particle size could only be determined forthe pH 5.5 sample. While the average particle diameter at pH 5.5 is verysimilar to the average particle diameter at pH 4.7, the increase inturbidity of the supernatant indicates the presence of some additionalsmaller nanoparticles, not present in the pH 4.7 samples.

FIG. 6 is a graph of the apparent viscosity of aqueous suspensions withPEG 300 and organic additives in 150 mM pH 4.7 acetate buffer with addedNaCl to 154 mM ionic strength. As shown in FIG. 6, upon addition of10-20% of an organic additive to at least 25% PEG300, the apparentviscosity of the suspension remained below 50 cP even for extremely highBSA concentrations greater than 300 mg/ml. Furthermore, as shown intable 3, all of these PEG300 plus organic suspensions reduce apparentviscosities to a range of 14-22 cP at a BSA concentration of 250 mg/ml.This viscosity range is approximately the same as for the higher ionicstrength samples (300 and 500 mM) discussed above (Table 4). ThesePEG300-organic samples gave the lowest intrinsic viscosity values(5.8-6.5) measured in this study (Table 3). All three samples with10-20% NMP added, gave particle diameters that were very close to theoriginal 20 μm milled particles. In addition, the soluble concentrationfor all three NMP samples was between 2.3-2.9 mg/ml, indicating thesuspension of greater than 99% of the BSA added at the 300 mg/ml level(Table 3). The sample with 20% ethanol added had the lowest solutionviscosity, giving the lowest suspension viscosity at 250 mg/ml of 14 cP(Table 3). The slightly higher soluble concentration of 4.8 mg/mlindicated that greater than 98% of the BSA added was suspended. However,this slight decrease in percent suspended was sufficient to decrease theaverage particle diameter to 7.4 μm. The slight decrease in particlesize could be seen in the SEM imaged.

All of the above samples contained milled model protein, BSA, without asugar lyoprotectant that can help stabilize a therapeutic protein at themolecular level against monomer aggregation. BSA is a fairly stableprotein and thus the molecular stability was not considered in thisstudy. However, milling can generate instabilities for many therapeuticproteins. {Maa} On the other hand, freezing and lyophilizing of proteinparticles has been shown to produce molecularly stable protein particleseven for submicron sizes. A very common lyoprotectant is trehalose.

FIGS. 7A and 7B are graphs that show the apparent viscosities of milledBSA and trehalose at various ratios in 150 mM pH 4.7 acetate bufferalong with the theoretical viscosity as calculated from theKrieger-Dougherty equation using the [η] of the pure milled particles ina) 25% PEG 300 20% Ethanol and b) 35% PEG 300 and 15% NMP. FIG. 7 showsimages of data of protein and BSA particles at ratios from 1:0.1 to 1:1by weight BSA to trehalose. At all points, the higher ratios oftrehalose to BSA increase monotonically the viscosity of a suspensionformed in either 25% PEG300 20% ethanol or 35% PEG300 and 15% NMP. Eventhough the trehalose will be soluble in the aqueous solvents with theadditives, it will still contribute additional excluded volume that isunavailable either to the solvent or the undissolved particles. Thisexcluded volume will therefore decrease the highest loading of particlespossible to maintain a given viscosity. The theoretical lines in FIG. 7were calculated using the same intrinsic viscosity for the respectivesuspensions without trehalose and assuming a maximum volume fraction of0.64.

Antibody therapeutics currently constitute a market size of $15 billiondollars annually, addressing needs in anti-cancer, anti-infective andanti-inflammatory diseases. As large protein molecules, these currentlyrequire direct injection (intra-venous or subcutaneous) for delivery.Since the required doses are quite large and frequently administered,this poses a considerable obstacle for drug delivery: to achievesubcutaneous delivery with a small bore syringe, the antibody must beformulated in a high concentration (>100 mg/ml), low volume (<1.5 ml),low viscosity (<100 cP) format. These specifications have been verydifficult to achieve with traditional solution formulations (in aphosphate buffer containing trehalose) but suspensions of lyophilizedantibody in aqueous solvents containing salts and precipitants toprevent dissolution represent a possible option. We have previouslydemonstrated the success of this approach with a model protein, BSA andhere present an extension of the technology to polyclonal antibodies.Here we present for the first time, evidence that immunoglublins can beprepared as aqueous suspensions in concentrations up to 200 mg/ml, withlow viscosity and no aggregation (96% monomeric protein).

IgG purified from sheep serum (Product No. 15131) was purchased fromSigma-Aldrich, Inc. α-α trehalose, polyethylene glycol with an averagemolecular weight of 300 (PEG 300), ammonium sulfate, USP grade ethanol,and n-methyl 2-pyrrolidone (NMP) were purchased from FisherChemicals.

After dissolving a known amount of the IgG in an appropriate amount of a20 mM pH 5.5 histidine buffer with α-α trehalose, samples were slowfrozen on a pre-cooled lyophilizer shelf at −40° C. The samples werethen lyophilized for 12 hours at −40° C. at 100 mTorr, followed by a 6hour ramp to 25° C. at 50 mTorr, and maintained for secondary drying at25° C. at 50 mTorr for at least an additional 6 hours. 1 mg of powderwas then weighed out and reconstituted at 1 mg/ml in 200 mM pH 7.0phosphate buffer for stability analysis by size exclusion chromatographyas described below.

An aliquot of a 5 mg/ml IgG solution was mixed with an equal volume of asecond aqueous solution containing additives for the purpose of loweringthe protein solubility. The precipitation of the protein wascharacterized by an increase in turbidity of the solution after 24 hoursat a wavelength of 350 nm. The solutions were all formulated in a pH 6.420 mM histidine buffer with added PEG300 and NMP. The turbidity of a 100μl aliquot of the final formulation was measured on a UV-transparent96-well plate using a spectrophotometer.

Aqueous-based solvent mixtures, without protein, containing varyingvolume percents of NMP, PEG 300, and ethanol and various molarities ofsodium chloride or ammonium sulfate salts were mixed to form uniformtransparent solutions. These solutions were buffered at varying ionicstrengths usings either a histidine or phosphate buffer at pH 6.4 (theisoelectric point of sheep IgG). Samples of the protein were thencompacted into 0.1 ml conical vials such that the powder weight waswithin 5% of the desired weight. The powder weight depended upon thefinal protein concentration and the excpient/protein ratio. A measuredamount of the prepared aqueous-based solvent mixture was added to theconical vial to form a suspension with a total volume of 0.1 ml. Themixture was stirred the tip of a needle to remove air pockets and toform the suspension with sufficient uniformity. Sonication was not used,nor was it needed. A drop of the uniform suspension was then placed on amicroscope slide to image the suspension and 10 μl of the suspension wasdiluted to 1 mg/ml in a 200 mM pH 7.0 phosphate buffer to measure theprotein monomer fraction by size exclusion chromatography as specifiedbelow.

The apparent viscosity of the IgG suspensions was measured as the timeto draw 50 μl of the suspension into a 25 gauge 1.5″ needle attached toa 1 ml tuberculin slip tip syringe. A conically shaped vial was used tominimize the sample volume given the cost of the protein. Videos of theconical vial containing the suspension were taken and the time to drawfrom a height 0.4″ from the bottom of the cone to a height 0.1″ from thebottom of the cone was measured using Image J software. The uncertaintyin the height was on the order of 1%. The time was measured to within0.05 seconds as the video was converted to an image stack with 20 imagesper second. Each measurement was made at least 3 times and averaged,while maintaining the suction force constant for each measurement byholding the end of the plunger at the 1 ml mark each time. In most casesthe reproducibility in viscosity was 10%. Previous work with suspensionsof model proteins found that the time to draw up a specified amount ofthe sample in a syringe was correlated linearly to viscosity. A maximumvolume of 10% of the cavity in the syringe was filled with suspensionfor the uptake. Consequently, the pressure was essentially constant andthe viscosity may be obtained from the Poiseulle equation. In this case,using standard solutions with various viscosities (pure DI water,ethanol, PEG 200, PEG300, PEG400 and benzyl benzoate) gave a linearcorrelation between the time to draw 0.05 ml from the conical vial tothe viscosity with an r² value greater than 0.99.

Samples for scanning electron microscopy (SEM) of the dry powders afterlyophilization were placed on adhesive carbon tape. Each sample was thengold-palladium sputter coated using a Cressington 208 bench top sputtercoater to a thickness of 15 nm. Micrographs were then taken using aZeiss Supra 40 VP scanning electron microscope with an acceleratingvoltage of 5 kV. Optical microscope images of a drop of the finalsuspensions on a glass microscope slide were taken using an MTI CCD 72(Dage-MTI, Michigan City, Ind.) camera attached to a Nikon Optiphot2-Pol(Nikon Instruments Inc. Melville, N.Y.) microscope.

Percent monomer of the initial solution, reconstituted powder and finaldiluted suspension was analyzed by using Tosoh Biosciences G3000SWXLsize exclusion column followed by a G2000SWXL size exclusion columnattached to Waters Breeze HPLC system containing a model 717plusautosampler, 2487 dual wavelength detector, and 1525 binary pump (WatersCorporation, Milford, Mass.). The prepared samples, reconstituted ordiluted to ˜1 mg/ml in 200 mM pH 7.0 phosphate buffer were filteredthrough a 0.22 μm Millex-GV filter to remove large aggregates prior toanalysis. The mobile phase consisted of a pH 7.0 200 mM phosphate bufferand 50 mM sodium chloride at a flow rate of 0.7 ml/min. The detectionwavelength was 214 nm. An injection volume of 20 μl of the ˜1 mg/mlprepared sample was used. The monomer eluted at approximately 21.5minutes, with the higher molecular weight aggregates eluting in the lastfew minutes before this, depending on their size.

FIGS. 8A-8E are SEM images of various frozen powders of TgG. FIG. 8A isa SEM image of 40 mg/ml IgG frozen at a 1:1 IgG to trehalose ratio. FIG.8B is a SEM image of 55 mg/ml IgG frozen at a 1:0.5 IgG to trehaloseratio. FIG. 8C is a SEM image of 25 mg/ml IgG frozen at a 1:0.5 IgG totrehalose ratio. FIG. 8D is a SEM image of 40 mg/ml IgG no trehalose.FIG. 8E is a SEM image of 20 mg/ml IgG at a 1:1 IgG to trehalose ratio.Large micron-sized particles of IgG stabilized by α-α trehalose weremade by lyophilization using a 1:1, 0.5:1, 0.25:1, or 0:1 ratio oftrehalose to IgG in a 20 mM pH 5.5 histidine buffer at various initialconcentrations between 20-80 mg/ml of IgG. SEM micrographs of the finaldried powder show large 10-100 μm particles with relatively few fineparticles (on the order of hundreds of nanometers) for the particlesfrozen at higher concentrations (40 to 80 mg/ml) for each trehalose toIgG ratio (FIGS. 8A and 8B), including the case with no trehalose (FIG.8D). The large particles are in contrast to the smaller web-likemorphology visible for the protein frozen at lower concentrations, 20and 25 mg/ml IgG, with high ratios of IgG to trehalose, 1:1 and 1:0.5respectively (FIGS. 8C and 8E). During freezing, a higher concentrationof protein leads to greater growth and thus larger final particles.

The relative stability from SEC was defined as the difference in percentarea of the monomer peak after reconstitution of the dry powder in pH7.0 phosphate buffer relative to the initial powder diluted in the samepH 7.0 phosphate buffer. This relative stability was at least 98.6% andoften higher. The stability was high even for the 40 mg/ml IgG powderfrozen without any trehalose, indicating cryoprotectant is not needed toachieve high stabilities as measured by this technique. However thisvalue of 98.6 is lower than that of all of the other examples in thetable that included trehalose. Thus, a cryoprotectant can be beneficialfor increasing the stability, and trehaolse was included.

Precipitation of 5 mg/ml IgG solution with various additives. Variousadditives can be added to decrease the solubility of the IgG asdescribed in detail above. We have confirmed this by observation ofprecipitation in a high molarity (1.5M) ammonium sulfate solution at anIgG concentration of 5 mg/ml (optical density not determined).

FIG. 9 is a graph of the optical density of the IgG with variousadditives totaling 50% of the solvent by volume to decrease thesolubility of the IgG as measured at 350 nm. The right hand side has theabsorbance of the 5 mg/ml concentration of IgG in the pH 6.4 20 mMhistidine buffer with no additional additive. As shown in FIG. 9, at aconcentration of 5 mg/ml IgG, the absorbance at 350 nm increases from˜0.05 for the pure protein solution at pH 6.4 to ˜0.6 for a 50% volumesolution of PEG300 at pH 6.4 (FIG. 9). For the IgG at this concentrationin a 50% volume solution of NMP at pH 6.4, the absorbance at 350 nm wassignificantly lower at ˜0.2 than for the case of 50% PEG300 (FIG. 9).The absorbance at 350 nm of mixed samples of PEG300 and NMP totally 50%by volume of the solvent, with at least 25% PEG300 were similar. Theabsorbance decreased slightly as the % NMP increased from ˜0.6 to 0.5for a 25% PEG300 and 25% NMP mixed solution. However, a much lowerabsorbance of ˜0.2 was observed for a 50% NMP solution, without any PEG.These experiments indicate that proteins precipitate with theseadditives even at low protein concentration of 5 mg/ml. Therefore, onlya small fraction of the protein will be dissolved with these additiveswhen the overall protein concentration is on the order of 200 mg/ml, atypical concentration for the injectable suspensions.

Suspension morphology as a function of particle size. In addition to theratio of trehalose to protein in the lyophilized particles, the size andsurface area of the particles, will vary the morphology and viscosity(syringcability) of the suspension. The optimum particle size containsparticles small enough to flow up the 25 gauge needle however largeenough to minimize the detrimental effects of hydration andelectroviscous forces on the viscosity. Furthermore, a decrease in thesurface area of the particles may decrease denaturation and aggregationof the protein.

FIG. 10A-10D are images of various suspensions of IgG. A) 200 mg/ml IgGsuspension made of 55 mg/ml IgG 1:0.5 IgG to trehalose ratio particlesin 20 mM pH 6.4 histidine buffer with 1.5M added ammonium sulfate salt.B) 200 mg/ml IgG suspension made of 55 mg/ml IgG 1:0.5 IgG to trehaloseratio particles in 50 mM pH 6.4 phosphate buffer with 50% PEG300. C) 200mg/ml IgG suspension made of 55 mg/ml IgG 1:0.5 IgG to trehalose ratioparticles in 50 mM pH 6.4 phosphate buffer with 35% PEG300 and 15% NMPby volume after 24 hours D) 200 mg/ml IgG suspension made of 20 mg/mlIgG with 1:1 IgG to trehalose ratio particles in 50 mM pH 6.4 phosphatebuffer with 35% PEG300 and 15% NMP by volume.

In FIG. 10A and FIG. 10B, a concentrated suspension is shown for twodifferent additives and an IgG concentration of 200 mg/mL initiallyafter forming the suspension. The suspensions were white and opaque. Thepath length was approximately 0.5 cm at the mid-point of the cone. Theparticles in a droplet of the suspension were further characterized withoptical microscopy in FIG. 4. Micron-sized particles are present in therange of a few microns to 10 micron, consistent with the dry initialparticles from the SEMs in FIG. 8. FIG. 10C illustrates a typicalexample of a small degree of settling of these suspensions in FIG. 10Aand FIG. 10B after 24 hours. In FIG. 10D, the initial concentration forlyophilization was much lower, 20 mg/ml, and the particles were muchsmaller as evident in SEM and by optical microscopy of the suspension.These smaller particles did not scatter light as strongly, and thesuspension appeared translucent, instead of white and opaque.

FIG. 11A-11E are microscope images of various suspensions of IgG. FIG.11A is an image of 200 mg/ml IgG suspension made of 55 mg/ml IgG 1:0.5IgG to trehalose ratio particles in 20 mM pH 6.4 histidine buffer with1.5M added ammonium sulfate salt. FIG. 11B is an image of 200 mg/ml IgGsuspension made of 55 mg/ml IgG 1:0.5 IgG to trehalose ratio particlesin 50 mM pH 6.4 phosphate buffer 50% PEG300. FIG. 11C is an image of 200mg/ml IgG suspension made of 55 mg/ml IgG 1:0.5 IgG to trehalose ratioparticles in 50 mM pH 6.4 phosphate buffer 35% PEG300 15% NMP. FIG. 11Dis an image of 200 mg/ml IgG suspension made of 20 mg/ml IgG 1:1 IgG totrehalose ratio particles in 50 mM pH 6.4 phosphate buffer 35% PEG30015% NMP. FIG. 11E is an image of 200 mg/ml IgG suspension made of 80mg/ml IgG 1:1 IgG to trehalose ratio particles in 50 mM pH 6.4 phosphatebuffer 35% PEG300 15% NMP. Table 5 illustrates IgG lyophilized powdersmade at various protein concentrations and trehalose ratios in a 20 mMpH 5.5 histidine buffer, characterized for the stability of the drypowder by size-exclusion HPLC.

Protein concentration Trehalose:IgG SEC (% (mg/ml) (wt.) ratio monomerof original solution) 20 1:1 99.1 25 0.5:1   100.1 40 0 98.6 40 1:1101.6 55 0.5:1   99.8 65 0.25:1   99.9 80 0 100.1

Table 6 illustrates 200 mg/ml IgG suspensions in a solvent containing35% PEG300, 15% N-methyl-2-pyrrolidone (NMP) by volume added to a 50 mMpH 6.4 phosphate buffer. (ND—not determined; NM—immeasurable).

Frozen IgG Concentration Trehalose:IgG (wt.) Viscosity SEC (% monomer(mg/ml) ratio in frozen powder (cP) of dry powder) 20 mg/ml IgG 1:1 ND102.6 40 mg/ml IgG 0  52 79.7 40 mg/ml IgG 1:1 194 98.0 55 mg/ml IgG0.5:1   104 97.1 65 mg/ml IgG 0.25:1   144 86.9 80 mg/ml IgG 0 NM ND

The largest particles were formed with pure IgG particles frozen at 80mg/ml as shown in FIG. 11E. The large particle size was caused by thehigh starting concentration during lyophilization. They were suspendedin the 35% PEG300 15% NMP solvent described in Table 2. The particlesize reached >50 micron, and therefore, the particles did not flowthrough a 25 gauge syringe. In contrast, all of the smaller particles inFIG. 11 were syringeable.

Table 8 illustrates IgG suspensions in various buffers with variousadditives screened for their viscosity and % monomer of the originalsample present. (ND—not determined; EtOH—ethanol,NMP—N-methyl-2-pyrrolidone).

IgG Trehalose:IgG SEC (% Suspension Frozen IgG (wt.) ratio monomerSuspension Suspension Concentration Concentraion in frozen Viscosity ofdry Additive Buffer (mg/ml) (mg/ml) powder (cP) powder) 50% PEG300 20 mMpH 7.4 100 40  1:1 46 ND histidine buffer 50% PEG300 50 mM pH 6.4 170 550.5:1 72 102.0 phosphate buffer 50% PEG300 50 mM pH 6.4 200 55 0.5:1 78ND 90 mM (NH₄)₂SO₄ phosphate buffer 40% PEG300 40 mM pH 7.4 100 40  1:143 ND 10% EtOH histidine buffer 35% PEG300 50 mM pH 6.4 200 55 0.5:1 92 93.6 15% EtOH phosphate buffer 30% NMP 20 mM pH 7.4 200 40  1:1 71 ND10% PEG300 histidine buffer 1.5M (NH₄)₂SO₄ 20 mM pH 6.4 200 55 0.5:1 12 97.8 histidine buffer 1.5M (NH₄)₂SO₄ 20 mM pH 6.4 300 55 0.5:1 99 101.2histidine buffer

Viscosities less than 100 cP are sufficient for syingeability through a25 gauge 1.5″ syringe. Syringeable viscosities were obtained forsuspensions at concentrations up to 200 mg/ml IgG (Tables 6-8). Theaddition of a cryoprotectant, such as trehalose, as seen previously forBSA powders {Miller aqueous BSA}, will increase the viscosity of asuspension (Table 6). This increase is caused primarily by the excludedvolume occupied by the cryoprotectant. For example, in Table 6, the 200mg/ml IgG suspension, with no trehalose has a viscosity of 52 cP asopposed to 104 for a 0.5:1 ratio of trehalose to IgG. The addition oftrehalose increases the total solute (trehalose plus IgG) concentrationto 300 mg/ml. Further increasing the total solute concentration to 400mg/ml by increasing to a 1:1 ratio of IgG to trehalose, raises theviscosity further to 194 cP. Thus, the potential need for acryoprotcctant to form stable protein molecules must be balanced againstthe increase in viscosity due to the excluded volume of acryoprotectant. To examine the relationship between the precipitationseen in the solubility study above and the viscosity of the suspension aseries of tests were run using the 55 mg/ml IgG 0.5:1 trehalose:IgGparticles. For the additive conditions in the first three entries inTable 7, high ODs were obtained at even the low protein concentration of5 mg/ml in the solubility determinations in FIG. 9. The vials for these150 mg/ml protein suspensions were white opaque as in FIG. 3. Thesesamples had measurable viscosities between 57 and 98 cP. For theadditive compositions in the last two entries with only 15 or 0% PEG,and the remainder NMP, the OD was much lower for the precipitationstudies at 5 mg/ml protein, indicating higher protein solubility. Forthese additive compositions and 150 mg/ml suspensions, viscosities wereeither very high, 287 cP, or not measurable, as a paste-like gel wasformed. Thus, the additive compositions that cause significant proteinprecipitation at 5 mg/ml in FIG. 9, also are beneficial for producinglower viscosities. As the ratio of dissolved protein to micron-sizedprotein protein particles goes down, the viscosity is decreased. Thisdecrease may be attributed to a reduction in solvation andelectrosviscous forces, although further characterization would beneeded to more fully describe the mechanism.

In Table 8, miscellaneous additive conditions beyond those in Tables 6and 7. The aqueous solutions contained pure salt, pure PEG300, andmixtures of salts, PEG300 and water-soluble organic additives up to atotal of 50% of the solution (Table 8). Syringeable aqueous-based IgGsuspensions were obtained in all of these cases with a 25 G 1.5″ needle.In each of these cases, micron-sized particles were formed given the IgGfreezing concentrations of 40-55 mg/ml. For all the suspensions in thistable, the presence of micron-sized particles was confirmed with theoptical microscope (select samples shown in FIG. 11). The high molaritysalt (1.5M) additive gave the lowest viscosity of 12.2 cP at an IgGconcentration of 200 mg/ml (Table 8). At an IgG concentration of 200mg/ml with trehalose, the next lowest viscosity was for the 50% PEG300sample at 79 cP (Table 8). Adding 15% NMP and decreasing the PEG300 to35% at a pH of 6.4, increases the suspension viscosity up to 104 cP(Table 8). A different organic additive with a similar dielectricconstant, ethanol, at the same 15% level, gave a slightly lowerviscosity at 92 cP (Table 4). As previously demonstrated for BSA, thevarious additive compositions that decrease the solubility of a proteinbelow 5 mg/ml can often give highly precipitated suspensions andviscosities less than 100 cP at IgG concentrations greater than 100mg/ml. Certain additive compositions allow such viscosities up to 200mg/ml protein, and it may be expected that even higher proteinconcentrations may be achieved by further optimization.

As mentioned in Table 5, powders containing protein highly stableagainst monomer aggregation were achieved with or even without trehaloseas a cryoprotectant. We also examined the protein stability afterforming the suspensions. For the final suspensions, 10 μl of thesuspension were diluted in pH 7.0 phosphate buffer necessary to give afinal TgG concentration of ˜1 mg/ml. The relative stability was measuredas the difference between the % monomer after redilution when comparedto the % monomer for the initial lyophilized particles uponreconstitution. For all of the examples in Tables 6-8, where trehalosewas present at 0.5:1 or higher and without organic solvent (NMP orethanol), the monomer was high, at least 97%. The % monomer was >100% insome cases, either because of experimental error or an actual increasein the monomer fraction relative to the as received starting bulkmaterial. The high stabilities for systems with high PEG levels was notunexpected as PEG is known to help maintain the thermal stability of aprotein. {Stevenson} High stabilities are shown in the last two rows ofTable 4 for the two cases with a high salt concentration (1.5M ammoniumsulfate). The high salt concentration produces very low proteinsolubilities and favors the presence of the micron-sized proteinparticles.

For the two studies without trehalose in Table 6 the protein aggregationwas significant. After the powder with no trehalose was suspended, SECshows approximately a 20% loss in % monomer to small aggregates (row 2in Table 6). A smaller decrease in the % monomer of ˜15% was seen forparticles with an insufficient ratio of trehalose (0.25:1 trehalose toIgG ratio) (Table 6). Thus trehalose at a ratio of 0.5:1 of trehalose toIgG was required to maintain stability of the protein in the suspension,despite the fact that trehalose had a small effect on stability for theinitial powers in Table 5.

The behavior is more complicated for the systems containing NMP and PEG.As shown in the first thre rows in Table 7, the protein stabilitydecreases with an increase in NMP concentration for a constant overalladditive concentration of 50%. However, at a higher 1:1 IgG to trehaloseratio with the 35% PEG 300, and 15% NMP, the initial monomer was 98%.Thus, the higher ratio IgG to trehalose may compensate for the higherdegree of organic additive (NMP) to maintain the stability.

The protein stability for the suspensions may be compared with those forprotein in buffer without the addition of agents to lower thesolubility. The power formed at 55 mg/ml initial protein (Table 5) wasadded to pH 6.4 buffer without any other additives. The resultingmixture was translucent and less turbid than in any of the entries inTables 5-8. Upon centrifugation at 16,100 g, a precipitate was formedwith a volume less than 10% of the total protein volume. Thus, most ofthe protein was dissolved. The % monomer was 70% for the same procedureas for the suspensions above. In contrast, the monomer % was 97.1 forthe same powder (55 mg/ml) at the 200 mg/ml level for an opaque whitesuspension of micron sized particles in Table 6. Thus, micron-sizedparticles of protein can be far more stable than proteins primarily inthe dissolved state at high concentrations.

Stable as measured by SEC, highly concentrated aqueous based suspensionsof a model IgG were created using particles that were frozen andlyophilized at high concentrations (40-55 mg/ml). This concentrationrange (40-55 mg/ml) made particles with a diameter of ˜10-100 μm thatwere found to be greater than 98% stable by SEC. By stabilizing theparticles with trehalose at a minimum ratio of 0.5:1 trehalose to IgG,the final suspensions were also found to preserve at least 92% of theoriginal monomer percent. The solubility of the IgG was lowered to lessthan 5 mg/ml in the aqueous-based solvent by adding high salt (1.5 Mammonium sulfate), PEG300 (50% of solvent by volume), or a combinationof PEG300 and ethanol or NMP (total 50% of solvent by volume). Theapparent viscosity through a 25 g 1.5″ syringe of the high saltsuspension, where the solvent viscosity is still ˜1 cP, was the lowestat approximately 12 cP for a 200 mg/ml stable (by SEC) IgG suspension.Overall, the stability of the model IgG and the low viscosities (lessthan 100 cP) through a 25 g 1.5″ needle obtained for highly concentratedsuspensions indicates a potential advancement in the subcutaneousdelivery of protein therapeutics.

The delivery of concentrated proteins and peptides in the range of 100to 400 mg/ml by subcutaneous injection through a 25 to 27-gauge needlebecomes feasible for a stable solution or suspension with a viscositybelow about 50 cP. Viscosities below this limit were achieved forsuspensions of milled lysozyme microparticles in benzyl benzoate orbenzyl benzoate mixtures with vegetable oils for up to 400 mg/mlprotein. The protein molecules were stable against aggregation for atleast 2 months and the solid particles in suspension were resuspendableafter being stored at room temperature for a year. Correlations betweenthe viscosity of the suspension and the volume fraction of particlesindicate that the main source of interaction between the particles wassimply due to the high concentration of particles with little effectfrom additional forces such as electrostatic repulsion, solvation of theparticles or deviations of the particle shape from a spherical geometry.In contrast these additional forces can cause large increases inviscosities for colloidal protein molecules in aqueous solutions. Thusthe lower solvent viscosities for highly concentrated proteinsuspensions relative to protein solutions may offer novel opportunitiesfor subcutaneous injection.

As used herein, “stable proteins” refer to proteins that do not showinstabilities such as denaturation or aggregation of the individualprotein molecules in the dissolved state. These instabilities can bemeasured by techniques such as optical turbidity, dynamic lightscattering, size-exclusion chromatography, analyticalultracentrifugation, and a protein dependant activity assays.

Solvents for use with the present invention include those in which thenon-aqueous suspensions that produced stable particles not change inparticle size over 2 months of storage with a protein solubility of lessthan 0.03 mg/ml. The solvent must also not cause an adverse affect onthe stability of the protein particles. Stability of protein particlescan be obtained for a non-aqueous solvent where the absorption of waterinto the particles in the suspensions is approximately equal to theabsorption of water into particles exposed to ambient air conditions atthe same relative humidity. In addition, a non-aqueous solvent shouldhave a low dielectric constant (less than 37.5) to prevent attractiveforces between the particles to cause caking of the particles at thebottom of the container causing the suspension to be unable to beresuspended.

The objective of this study was to produce a highly concentrated proteinsuspension for delivery of high dosages of monoclonal antibody bysubcutaneous injection via a 25- to 27-gauge syringe. Suspensions oflysozyme particles smaller than 37 μm with concentrations from 50 to 300mg/ml were formulated with a 50/50 volume mixture of thepharmaceutically acceptable solvents safflower oil and benzyl benzoate.This solvent mixture was within the approved range published on theFDA's Inactive Ingredients List.¹⁸ While benzyl benzoate is less viscousthan safflower oil, which facilitates formation and injection of thedispersions, it is not currently accepted for injection by the FDA as apure solvent; however, indications of lack of toxicity suggest it may beapproved in the future. The apparent viscosities for formulations in theapproved solvent mixture as well as pure benzyl benzoate will be shownto be in an acceptable range up to a concentration of at least 300 mg/mland to correlate with theoretical viscosities of suspensions accordingto the Krieger-Dougherty equation. The necessity to obtain a uniformdose is addressed through measurements of the settling rate andconfirmed by concentration measurements of aliquots of the suspension.Colloidal stability of the particles and the stability of the proteinmolecules are addressed by measurements of particle and proteinaggregation over time and are further confirmed by analyzing themoisture content in the various suspensions.

Lysozyme in lyophilized powder form was purchased from Sigma ChemicalCompany (St. Louis, Mo.). ACS grade acetonitrile and USP grade ethanolwere used as received from Fisher Chemicals (Fairlawn, N.J.). Food gradeolive oil and safflower oil were purchased for initial tests from thegrocery store. Benzyl benzoate was obtained from Acros Organics (NewJersey) and N.F. grade ethyl oleate from Spectrum Chemical Corp.(Gardena, Calif.).

Lysozyme powder as received was dry milled with a porcelain mortar andpestle for several minutes. The milled powder was then sieved through anumber 400 mesh and particles smaller than 37 μm were collected. Sampleswere then weighed and added to a measured amount of benzyl benzoate or apremixed 50/50 volume mixture of benzyl benzoate and safflower oil. Eachvial was then shaken by hand to disperse the powder evenly through thesuspension.

Particle size was measured by multiangle laser light scattering with aMalvern Mastersizer-S (Malvern Instruments, Ltd., Worcestershire, UK).The milled and sieved powder sample size was measured as a suspension inacetonitrile in a large recirculation cell (˜500 ml) and immediatelyafter being added to ethanol in a small batch cell (Malvern,Worcestershire, UK, ˜15 ml). In each case the obscuration during themeasurement was between 10-15%. After storing the suspensions for 2months at room temperature, the particle size was measured againimmediately after shaking and diluting the sample in ethanol in thesmall batch cell.

The viscosity was measured as the time to draw 1 ml of the sample into asyringe with a 25 g ⅝″ or 27 g ½″ needle. Each measurement was made atleast 3 times and averaged. Liu et al. found this measurement time to becorrelated linearly to viscosity. From known viscosities of each pureliquid, benzyl benzoate, ethanol, ethyl oleate and olive oil, thecorrelation between the time to draw 1 ml of solution and the viscositywas found for each needle size to give an r² value greater than 0.999.The apparent viscosity of the suspensions in each pure solvent wascalculated from these correlations and the values for the two separateneedle sizes were averaged to give a final average apparent viscosity ofeach sample. Additional samples of the solvent mixture of benzylbenzoate and safflower oil were made at 10, 20, 30, 40, 50, 60, 70, 80,and 90 percent benzyl benzoate by volume. The viscosity of each samplewas calculated as described above.

The settling rate of the particles in the solvents was measured byshaking up the suspension in a test tube 13 mm in diameter. Pictureswere taken with a standard digital camera after 10, 30, 60, 90, 120,150, 180, 210, 240, 1200, and 1440 minutes. To measure the finalsettling volume of the samples, the vials containing the suspensionswere left undisturbed for 4 months and images of the settled suspensionwere taken. All images were analyzed using ImageJ software for thedistance from the meniscus to the settling front. The maximum volumefraction for the settled suspension was defined by dividing the volumefraction of particles in the overall suspension by the ratio of thevolume containing particles after settling for 4 months to the overallvolume.

The concentration of lysozyme in an aqueous solution was measuredfollowing the protocols for the Micro BCA Protein Assay. Each sample wasmeasured in triplicate with relative standard deviations (% RSD) lessthan 2% in a General Assay 96 well plate (see statistical analysisbelow). The absorbance was measured at 562 nm in a spectrophotometer. Astandard curve of untreated lysosyme was prepared at concentrationsbetween 2 and 30 μg/ml.

Partitioning and dissolution of lysozyme from the concentratedsuspension was measured in a pH 7.4 potassium phosphate buffer. The USPpaddle method was used with a VanKel VK6010 Dissolution Tester with aVanderkamp VK650A heater/circulator. 900 ml of dissolution media waspreheated in large 1 L capacity dissolution vessels (Varian Inc., Cary,N.C.) to 37° C. A sample of the concentrated suspension giving a totalof 18 mg of lysozyme was added. At time increments of 2, 5, 10, 20, 40,60, 120 and 240 minutes, 1 ml samples were taken and analyzed using theMicro BCA protein analysis mentioned earlier. 0.1 ml of the concentratedlysozyme suspension was added to a test tube with 4 ml of DI water. Thismixture was then gently mixed and left for 3 days for the protein topartition to the water phase. The water phase was then separated and asample was diluted and tested for concentration using the Micro BCAProtein Assay as mentioned above. The remaining aqueous solution wasdiluted to a concentration of 1 mg/ml. This solution was tested foroptical density using at least 3 300 μl aliquots in a 96-well plate andanalyzed using the μQuant spectrophotometer at 350 nm. A standardlysozyme aqueous solution was made at 1 mg/ml concentration and exposedto the pure benzyl benzoate solvent and the benzyl benzoate andsafflower oil solvent mixture for 3 days and measured as the standardfor oil-water interface induced aggregation of the protein. The aqueoussolution without being exposed to any organic solvent was also measuredimmediately after it was made and used as the standard absorbance forall measurements.

Three separate 0.1 ml aliquots of the resuspended concentrated lysozymesuspensions were added to test tubes with 8 ml of DI water. Thesemixtures were then gently mixed and left for 1 day for the protein topartition to the water phase. The aqueous phase was then separated anddiluted to a theoretical concentration of 20 μg/ml if 100% of theprotein partitioned. The actual concentration was then analyzed usingthe Micro BCA protein assay mentioned above.

Karl Fischer Moisture Analysis. After being stored for four months, asample of 0.1 ml of the redispersed concentrated suspension was insertedusing a 19-gauge needle through the septum of the titration cell of anAquatest 8 Karl-Fischer Titrator (Photovolt Instruments, Indianapolis,Ind.). Each suspension, pure benzyl benzoate and the benzyl benzoate andsafflower oil solvent mixture was measured in triplicate and averaged.

Polarity Determination. An aliquot of the suspension was diluted withthe solvent until individual particles were visible on a slide throughan optical microscope (Bausch & Lomb, 10× magnification).Microelectrophoresis was used to determine if a charge was present onthe particles. The diluted particle dispersion was placed between twoparallel wire electrodes (0.01-in. diameter stainless steel 304 wire,California Fine Wire) spaced 1 mm apart. The electrodes were secured toa glass microscope slide and observed by optical microscopy. A potentialof 10-100 V was applied with the polarity of the electrodes switchedevery 15-60 sec.

Samples for protein concentration, Karl Fisher moisture analysis,suspension uniformity, optical density, and rate of lysozymepartitioning to water were measured in triplicate to determine the mean,standard deviation and the relative standard deviation (std. dev./mean).

For lysozyme particles that were milled by mortar and pestle and sievedthrough a number 400 sieve, the average particle size was approximately20 μm, according to light scattering measurements (FIG. 12). A minorsecondary submicron peak was also visible in all measurements. However,since the 15 ml small batch cell is only calibrated for particle sizesdown to 500 nm, this peak was not included in the analysis. The vialcontaining the particles and solvent is then shaken by hand and theparticles disperse to form a uniform suspension (FIG. 13B). When theparticle suspensions are allowed to sit undisturbed, they settle slowlyenough to remain partially suspended even after 24 hours (FIG. 13C) andthe highly concentrated suspension takes up a significant portion of thevolume even after 2 months (FIG. 13A).

Viscosity of Solvent Mixture and Suspensions. Using the knownviscosities of pure solvents, a correlation between the time to draw 1ml of the sample and viscosity was generated. This type of correlationhas been described by Shire and coworkers on the basis of theHagen-Poiseuille equation⁵⁻⁷

$\begin{matrix}{{\langle v\rangle} = {\frac{R^{2}}{8\eta}\left( \frac{{\Delta \; P}}{L} \right)}} & {{Eq}.\mspace{14mu} 2}\end{matrix}$

where υ is the velocity, R is the inner radius of the needle, η is theviscosity, and ΔP/L is the average pressure drop over the length of theneedle. Ensuring that the average pressure drop over the length of theneedle remains constant for each sample by maintaining the same suctionpressure inside the syringe, the inverse of the velocity of the fluidmultiplied by the cross-sectional area gives the time to draw up aspecified volume of liquid, in this case 1 ml. This time is proportionalto the viscosity as shown by the Hagen-Poiseuille equation.

The measured viscosities of the solvent mixtures of benzyl benzoate andsafflower oil are shown in FIG. 14. In this case, since the minimum andmaximum values are the for the pure solvents, the generalized mixingrule should follow the form

$\begin{matrix}{{f\left( \eta_{m} \right)}_{L} = {\sum\limits_{i}\; {x_{i}{f\left( \eta_{i} \right)}_{L}}}} & {{Eq}.\mspace{14mu} 3}\end{matrix}$

where η_(m) is the viscosity of the mixture, i is the number ofcomponents, x_(i) is the liquid volume, weight, or mole fraction, andη_(i) is the viscosity of the i^(th) component. f(η)_(L) can beln(η_(L)), 1/η_(L), or another typical equation.²⁰ In this case, thecorrelation most closely associated with the experimental results waswhen f(η)_(L) was ln(η_(L)). This theoretical result is shown by thedotted line in FIG. 14.

The apparent viscosities of the suspensions with increasingconcentration were measured for both the pure benzyl benzoate system andthe solvent mixture of 50/50 benzyl benzoate and safflower oil. Theresulting viscosities, averaged from the measurements using two syringesizes (left y-axis), and the time to draw 1 ml from the 25-gauge syringe(right y-axis) were plotted against the concentration of lysozymeparticles (FIG. 15). The correlation of the apparent viscosity with thefree solvent volume fraction was modeled using the Kreiger-Doughertyequation

$\begin{matrix}{\frac{\eta}{\eta_{o}} = \left\lbrack {1 - \left( \frac{\varphi}{\varphi_{\max}} \right)} \right\rbrack^{{- {\lbrack\eta\rbrack}}\varphi_{\max}}} & {{Eq}.\mspace{14mu} 4}\end{matrix}$

where η is the apparent viscosity of the dispersion, η_(o) the solutionviscosity, φ the volume fraction of particles, φ_(max) the maximumpacking fraction, and [η] the intrinsic viscosity. φ_(max) wasapproximated after gravitational settling of the particles over 4months. It was approximately 0.50 for the pure benzyl benzoate solventsolution and 0.52 for the benzyl benzoate and safflower oil solvent forlow shear rates Using these values, the intrinsic viscosity of thesuspension, [η], was determined to be 2.7 for the pure benzyl benzoatesuspensions and 2.3 for the benzyl benzoate and safflower oilsuspensions.

The stability of the particles in suspension was measured by numerousdifferent techniques. First the settling rate was calculated andcompared to the theoretical Stokes settling rate

$\begin{matrix}{U_{s} = \frac{2\; r^{2}{\Delta\rho}\; g}{9\eta_{o}}} & {{Eq}.\mspace{14mu} 5}\end{matrix}$

where r is the radius of the particles, Δp is the difference indensities between the solvent and the particles, and g is accelerationdue to gravity. For a high concentration of particles, the particlecrowding will reduce the settling rate to yield

U=U _(s)(1−φ)^(6.55)  Eq. 5

This modified Stokes settling rate and the experimentally measuredvalues were found to be within a factor of two for most concentrationslower than 300 mg/ml as shown in Table 1. However, for a concentrationof 400 mg/ml the values are an order of magnitude lower than thepredicted rate (Table 9).

TABLE 9 Comparison of experimental settling rates and settling ratesquantified by the modified Stokes settling equation accounting forparticle interactions (eq. 4, 5). volume Modified fraction ofExperimental Stokes particles settling settling Formulation (Φv) rate(cm/min) rate (cm/min) 50 mg/mL Lys 0.03515 0.0308 0.0141 in 50/50Safflower Oil and Benzyl Benzoate 100 mg/mL Lys 0.0703 0.0220 0.0111 in50/50 Safflower Oil and Benzyl Benzoate 200 mg/mL Lys 0.1406 0.00530.0066 in 50/50 Safflower Oil and Benzyl Benzoate 300 mg/mL Lys 0.21090.00071 0.0038 in 50/50 Safflower Oil and Benzyl Benzoate 50 mg/mL Lys0.03515 0.0651 0.0373 in Benzyl Benzoate 100 mg/mL Lys 0.0703 0.02740.0293 in Benzyl Benzoate 200 mg/mL Lys 0.1406 0.0087 0.0175 in BenzylBenzoate 400 mg/mL Lys 0.2812 0.00052 0.0054 in Benzyl Benzoate

The suspension uniformity was further quantified by the percentextracted into an aqueous phase. Initially the rate of partitioning ofthe lysozyme into the aqueous phase was determined to requireapproximately 60 minutes. Three aliquots from the resuspended sampleswere placed in separate test tubes and allowed to partition to theaqueous phase for 1 day to ensure complete partitioning. The aqueousphase was then diluted approximately 1000 times, and the concentrationof protein was measured. The results show that even with a small volume(8 ml) of aqueous phase exposed to 0.1 ml of the concentratednon-aqueous suspension at least ¾ of the protein partitions into theaqueous phase in 24 hours (Table 10). The % RSD values were typicallybelow 5% indicating reasonable uniformity of the protein particleswithin the redispersed suspension. The % RSD was slightly larger for thehighly concentrated 300 mg/ml sample in the mixed solvent.

TABLE 10 Percent of sample recovered in aqueous phase and % relativestandard deviation (% RSD) of 3 samples. Concentration % recovered inSolvent (mg/ml) aqueous % RSD Safflower Oil and Benzyl 50 77.2% 2.21%Benzoate Safflower Oil and Benzyl 100 78.5% 6.61% Benzoate Safflower Oiland Benzyl 200 85.8% 3.20% Benzoate Safflower Oil and Benzyl 300 76.2%9.45% Benzoate Benzyl Benzoate 50 85.2% 1.52% Benzyl Benzoate 100 96.9%3.46% Benzyl Benzoate 200 92.2% 3.65% Benzyl Benzoate 400 81.2% 4.29%

The aggregation of the particles exposed to the non-aqueous solvent wasalso tested to ensure that growth of the particles is not occurring at asufficient rate to jeopardize the storage of the samples. The originalparticle size was measured via light scattering immediately after theparticles were sieved and resuspended in both acetonitrile, wherelysozyme is very insoluble, and ethanol, where lysozyme is slightlysoluble. The uniformity of the two measurements ensures that the timescale of the measurement is much quicker than the time scale of growthof the particles in ethanol (FIG. 12). Following 2 months of storage,the samples were diluted in ethanol and immediately tested. The particlesize was found to be essentially constant during storage (FIG. 12). Thevisual inspection of one formulated suspension after storage for a yearand redispersion by shaking confirms that the particles could beredispersed.

The potential effect of electrostatic repulsion on the particlestability was tested. However the lysozyme particles did not displayorganized movement when the voltage was changed from 10 to 100 for twoelectrodes spaced 1 mm apart in the benzyl benzoate solvent.

Protein aggregation was investigated by measuring the optical density onsample aliquots that partitioned from the organic to the water phase.The protein was diluted to a concentration of 1 mg/ml. Additionallysozyme samples in an aqueous solution at the same concentration wereexposed to the solvent to measure the effect of the oil-water interfaceon aggregation. All these solutions were checked for large proteinaggregates by comparison with a fresh lysozyme solution at the sameconcentration. No aggregates were found since the absorbance at 350 nmwas within 1% for the standard and all samples and therefore within theerror of the study.

Quantification of the moisture content may be used to determine the freeand bound water in the suspension. The moisture content was measured foreach suspension after being exposed to atmospheric conditions for 2months. The linear correlations found between the moisture content andsuspension concentration indicates that the moisture is directlyassociated with the protein (FIG. 16). The benzyl benzoate solventcontains an average of 20 μg of water per 0.1 nil of solution orapproximately 0.02% by weight. The safflower oil and benzyl benzoatemixture contains approximately 74 μg of water in the same volume sampleor approximately 0.074%. The sample with the highest concentration ofprotein in benzyl benzoate, 400 mg/mL, contained the most moisture, anaverage of 4450 μg of water per 0.1 ml of solution giving an absolutemaximum concentration of 4.5% by weight of the suspension after beingstored for 2 months.

In addition to protein and particle stability, the other key criterionis that the suspension's apparent viscosity must be low enough forinjection through a syringe. For subcutaneous delivery, 50 cP is anappropriate maximum viscosity where it will take approximately 20seconds for 1 ml of the suspension to be expelled via a 26-gaugesyringe. From FIG. 15, the highest apparent viscosity measured wasapproximately 50 cP, where it took approximately 55 seconds to draw 1 mlinto a 25-gauge syringe. The disparity in the times measured reflectsthe smaller suction force to draw the volume into the syringe relativeto the force needed to expel the solution from the syringe. In addition,these concentrated suspensions are considered to be shear thinning sincethe flow of the suspension will produce a more favorable rearrangementof the particles. For example, as the shear rate increases, sphericalparticles that were initially randomly packed (φmax=0.64) become moreordered and pack tighter, giving a higher maximum packing fractionaround 0.71. As a result, at the higher shear rate associated withexpelling the volume, the measured apparent viscosity of the suspensionswill decrease and will remain below the maximum for potentialsubcutaneous delivery.

Further analysis of the viscosity of the suspensions using theKreiger-Dourghety equation can give an indication of the effect ofinterparticle forces in the suspension. The initial equation for theviscosity of a dilute suspension derived by Einstein takes into accountthe particles and assumes that the particles are solid spheres and theirconcentration is low enough for the particles to be treated individually(φ<0.1). This gives a first order equation where the volume fraction ofthe particles is related to the viscosity ratio of the suspension overthe solvent with a slope of 2.5. In more general terms, this slopesignifies the increment of viscosity due to the addition of dispersedparticles and is also called the intrinsic viscosity, [η]. For moreconcentrated suspensions, accounting for particle crowding and themaximum packing fraction of the suspension (φmax) results in theKrieger-Dougherty Equation (Equation 5).31,32 In this case, theintrinsic viscosity term can vary from the Einstein coefficient value of2.5 depending on the effects of solvation, varying shapes, andelectrostatic forces as well as the shear rate. Since the values for theintrinsic viscosity of the benzyl benzoate and the benzyl benzoate andsafflower oil mixture suspensions are close to the original Einsteinderived 2.5, the effects of solvation, varying shapes, and electrostaticforces may be considered to be negligible assuming the shear rate is lowand can be approximated as zero. The lack of electrostatic effects wasnot surprising given the tendency for ion pairing in the solvent with adielectric constant of only 4.8. This is further confirmed with the lackof electrophoretic mobility measured above. If the particles aresolvated by the solvent the volume fraction would increase by

$\begin{matrix}{\frac{\varphi_{solvated}}{\varphi_{dry}} = \left\lbrack {1 + {\left( \frac{m_{1,b}}{m_{2}} \right)\left( \frac{\rho_{2}}{\rho_{1}} \right)}} \right\rbrack} & {{Eq}.\mspace{14mu} 7}\end{matrix}$

where m_(1,b) is the mass of the bound solvent, m₂ is the mass of theparticle, ρ₂ is the density of the particle and ρ₁ is the density of thesolvent. In the case of the Krieger-Dougherty equation, this increase isabsorbed into the [η] term, increasing the measured intrinsic viscosity.The deviations of the particles from spherical shape has a strong effecton the maximum packing fraction and on the intrinsic viscosity. Forexample, glass fibers of varying axial ratios, 7, 14 and 21 increase inintrinsic viscosity from 3.8 to 5.03 to 6.0, respectively, and decreasein maximum packing fraction from 0.374 to 0.26 to 0.233.

Since a large macromolecule, such as a monoclonal antibody in solution,can be approximated as a small colloid, similar viscosity analysis canbe conducted. In this case, previously published values for the increasein viscosity of a solution containing a monoclonal antibody at varyingconcentrations was used giving a final value of [η] of 45 from analysisusing the Krieger-Dougherty equation (Table 11). However, the analysisof protein solutions is typically done using mass concentrations (g/ml)rather than volume fractions, leading to values of the intrinsicviscosity in units of cm3/g and slightly different derived higher orderrelationships between the viscosity of a protein solution and theaqueous solvent. A hard quasispherical model

$\begin{matrix}{\frac{\eta}{\eta_{o}} = {\exp\left( \frac{\lbrack\eta\rbrack c}{\left( {1 - {{\frac{k}{\upsilon}\lbrack\eta\rbrack}c}} \right)} \right)}} & {{Eq}.\mspace{14mu} 8}\end{matrix}$

where c is the mass protein concentration, k is a crowding factoraccounting for high concentrations of the protein and υ is the Simhaparameter accounting for the change in shape from a sphere, is derivedfrom the same power series as the Krieger-Dougherty equation; however,using concentration rather than volume fraction as the x component. As aresult, it leads to a value of 6.9 cm³/g for the intrinsic viscosity ofa monoclonal antibody and a κ/υ value of 0.533. For various proteins,the value of the intrinsic viscosity varies from approximately 2.7 forlysozyme to over 200 cm³/g. This model has been shown to accuratelypredict the viscosities of hemoglobin, bovine serum albumin, and twovarious monoclonal antibodies where long range electrostatic forces werefound to play a negligible role in the viscosity versus concentration.Even for lysozyme, a protein with a very small axial ratio, 1.5, thismodel shows a dramatic increase in viscosity around a concentration of300 mg/ml (FIG. 15). Therefore, for various monoclonal antibodies, BSA,and hemoglobin that can described by the hard quasispherical model andhave higher axial ratio than lysozyme (eq. 8), the rapid increase inviscosity will be more dramatic and occur at a lower concentration. Forthese solutions, the viscosity increase is due not only to particlecrowding but also to the excluded volume effects of solvation anddeviation of the shape from a sphere. This strong deviation is not seenfor particles in suspension at the same concentrations because they arefairly spherical, are not hydrated by the solvents, and the density of aprotein is typically around 1.35 g/cm³ leading to a lower volumefraction for the respective concentration.

TABLE 11 Comparison of the two solvent systems of suspensions and thehigh concentrated solution monoclonal antibody for the exponents for theKrieger-Dougherty equation, the experimental maximum packing fraction,and intrinsic viscosity. Exponent for Krieger- Maximum volume IntrinsicDougherty equation packing fraction viscosity Solvent system −[η]Φ_(max)Φ_(max) [η] Benzyl benzoate −1.362 ± 0.09 0.50 2.7 suspension 50/50Safflower −1.149 ± 0.06 0.52 2.3 Oil and Benzyl Benzoate suspension Mab1solution −45.27 ± 0.61 1 45.3 in aqueous solution

The viscosities of concentrated suspensions up to 300-400 mg/ml ofmilled particles of the model protein, lysozyme, were small enough forsubcutaneous injection through a 27-gauge needle. The protein moleculeswere stable against aggregation and the particle size did not vary forat least 2 months when stored at atmospheric conditions. The apparentviscosity was correlated with volume fraction at all conditionsaccording to the Krieger-Dougherty equation. Full settling of theparticles was found to take well over 24 hours, which gave sufficienttime for a uniform aliquot to be taken and analyzed. The resultsindicated sufficient suspension stability to allow for accurate dosing,either for, single injection or multiple injection applications.Overall, the colloidal stability and dose uniformity of the lysozymemicroparticle suspensions, along with the acceptably low viscosities,indicates a potential advance for subcutaneous delivery of therapeuticproteins. For highly concentrated proteins in solution, various forcesincluding electrostatic repulsion, solvation forces, and deviations ofthe particle shape from a spherical geometry can cause large increasesin viscosity, whereas these forces have almost negligible effects forthe current protein suspensions, resulting in much lower viscosities.

The objectives of this example are to: (1) use various particleengineering techniques to produce protein particles to form suspensionsin nonaqueous and aqueous solvents, (2) to find efficient ways to formthe particles in vials or transfer the particles to vials, (3) todetermine the particle size, colloidal stability and viscosity of thesuspensions, and (4) determine the stability of the protein moleculeswith regard to denaturation and aggregation. The relevant particleengineering techniques include milling, spray drying, precipitation, andthin film freezing. The feasibility of delivering proteins and peptidesby subcutaneous injection depends on formulating a sufficiently lowviscosity product that is syringeable through a 25 to 27-gauge needlebut contains the necessary high concentration of the active protein orpolypeptide to give a full dose in less than 1.5 ml of volume. Desiredviscosities have been made with protein suspensions with up to 400mg/mL.

Of primary importance in the formulation of a stable suspension systemthat contains a high concentration of the monoclonal antibody and asufficiently low viscosity is the formation of stable appropriatelysized monoclonal antibody particles. Previously, a suspension wasformulated with ˜10-20 μm milled lysozyme.1

In addition to milling, protein particles may be formed with thin filmfreezing (TFF), previously disclosed to produce stable nanoparticles ofproteins2, to produce similar micron-sized particles, depending upon thefeed concentration. Since the monoclonal antibody will come in solutionform, TFF will be a significantly less destructive process, causing onlypotential freezing stress whereas lyophilization followed by milling andsieving the particles will expose them to freezing, heating andmechanical stresses which can lead to denaturation. Proposed work willinclude tuning the TFF process to produce the specified particle size byaltering the feed concentration within the solubility limits of the Maband adding cryoprotecting sugars such as trehalose, varying the solvent(currently pure DI water with a buffer) to include a percentage ofethanol and various buffers and other excipients necessary for asubcutaneous injection, and examine freezing directly into appropriatevials for subcutaneous injection formulation.

Various techniques will be utilized to transfer the protein particlesinto the vials. The goal will be to simplify processing steps and tomaintain sterile conditions. The first method is to transfer lyophilizedpowder to the vials as a solid. A second method is to transfer theprotein to the vials while still in the frozen state. Particle size datawas obtained to demonstrate each of these methods using the TFF process(Table 12).

TABLE 12 Comparison of particle size distributions, measured bymulti-angle light scattering of protein particles suspended inacetonitrile, from different concentration distributions of TFF. Theparticles were transferred to vials either after lyophilization or afteronly freezing. Particle Size Sample Freezing Method (dV10, dV50, dV90)High concentration BSA Frozen in vial (18.93, 32.88, 53.07) Highconcentration BSA Separated after (0.32, 17.95, 45.07) lyophilizationHigh concentration LYS Separated after (0.23, 13.60, 31.30) primarydrying lyophilization High concentration LYS Separated after (0.31,13.17, 28.57) freezing Low concentration LYS Frozen in vial (0.12, 0.39,2.27)

Once appropriate stable particles are made, an appropriate solventsystem must be found. In this case, both aqueous and non-aqueous basedsystems will be analyzed. A stable, low viscosity formulation of milledlysozyme in the pure non-aqueous solvent benzyl benzoate and the solventsystem benzyl benzoate and safflower oil have been previously analyzedland will be analyzed further in a suspension containing the Mabparticles described above. To overcome the instabilities of the proteinor polypeptide in a highly concentrated aqueous solution and theincrease in viscosity caused by soluble aggregates and hydration, themain objective of this study was to formulate a protein suspension in anon-aqueous solvent or solvent mixture using the model protein lysozymethat is syringeable through a 27-gauge needle and at a concentrationgreater than 100 mg/ml. The suspensions formulated were found to remainsyringeable up to at least a concentration of 300 mg/ml. Protein andparticle stability remained for at least 2 months indicating apotentially stable protein product. Protein particles in suspension werealso found to be resuspendable after being stored at room temperaturefor a year. Correlations between the viscosity of the formulation andthe increasing volume fraction of particles indicates that the mainsource of interaction between the particles is due to particle crowdingand no additional forces such as electrostatic repulsion, solvation ofthe particles from the solvent, or deviations of the particle shape fromspheres are used to maintain the stability of the suspension.

However, the main focus will be on aqueous solvents, whereas thenonaqueous solvents will be of secondary interest. An aqueous solventsystem is slightly more complicated to formulate and must include: (1)an agent to decrease the solubility of the Mab in water (if solubilityis greater than 20 mg/mL); (2) an antifoaming agent to prevent theconcentrated suspension from producing a foam when the solvent is added;and (3) additional excipients to maintain the stability of the Mab andthe final suspension product for a sufficient period of time.

Agents that can be added to decrease the solubility of a protein inwater include high concentrations of salts such as sodium sulfate andammonium sulfate, complexing agents including zinc, water solublepolymers such as PEG, various organic solvents such as ethanol and othersurfactants such as Tween 20. Addition of salts increases the ionicstrength of the solution which decreases the solubility of thehydrophibic groups of a protein. However for subcutaneous injectabledelivery, a solution that is not isotonic will increase the pain uponinjection, potentially leading to noncompliance. Therefore, alternativesto decrease the solubility with other additives can be advantageous. Apolyethylene glycol (PEG) and water mixture has also been demonstratedto that similar “salting out” effects of salt and are more readilyallowed in injectable formulations. Different molecular weight PEGs willbe tried since lower molecular weight PEGs can be formulated to muchhigher concentrations for additional solubility decrease however highermolecular weight PEG have been found to increase the thermal stabilityof a protein. Tween 20 at a 1%(w/v) concentration has been demonstratedto cause precipitation of the hydrophobic protein Humicolalanuginosalipase5 which may prevent solubilization of the added Mab particles insuspension and create another alternative way to formulate thesuspension. Additional excipients, such as water soluble non-aqueoussolvents, where the Mab is only slightly soluble will also be triedincluding ethanol, propylene glycol, and dimethylacetamide.

Successful higher concentration formulations of low viscosities withsalt and PEG formulations have been made in aqueous media (Table 13).Initial particle size measurements as well components are alsodisclosed. These formulations have been found to contain particlestability for at least 1 hr (FIG. 17); long time stability has not beeninvestigated. Preliminary data indicate that the viscosities of some ofthe aqueous suspensions were sufficiently low for 25 gauge needles.

TABLE 13 Protein used, concentration, substance added to decreasesolubility, preliminary viscosity measurement, preliminary particle sizefor successful aqueous based formulations. Agent to Protein and decreaseprotein Viscosity concentration solubility measurements Particle size150 mg/ml PEG 300 57.5 cP (0.40, 13.98, 26.82) BSA aqueous solution 250mg/ml Salt aqueous  5.7 cP (0.27, 10.90, 26.91) LYS solution

Foaming of aqueous dispersions of proteins has been observed in a 1.0Msodium sulfate aqueous solvent suspension when the liquid was added tothe dried TFF particles. Foam stability of an aqueous suspension ofproteins and fat particles was reduced significantly with the additionof the different Span excipients where it was hypothesized to increasebubble coalesance. A trial using SPAN® 80 successfully broke the foamwhen added to a concentrated dispersion of BSA in a 1.0M Sodium Sulfatesolution, leaving a low viscosity, highly concentrated suspension. Othersurfactants will be used both in the particle formation and in the finalformulation stage to resolve the foaming issue. In addition, otherexcipients must be added to complete the formulation of apharmaceutically acceptable suspension to be administeredsubcutaneously. To stabilize the protein, surfactants such as Tween 20and Tween 80 can be added if necessary. To create a final aqueousdispersion, buffering agents, antioxidants, and antimicrobial agents maybe added.

Final formulated suspensions will then be tested for viscosity using thetime to draw 1 mL of solution up a syringe with a 25- then 27-gaugeneedle. As demonstrated previously, the viscosity approximated with thismeasurement is reasonable with by both theoretical and previous results(above). In addition, settling rate measurements, images taken byoptical microscopy, particle size measurements by multi-angle laserlight scattering and electrophoretic mobility measurements will be usedto characterize the stability of the suspension. The settling rate canbe useful to determine the uniformity of the suspension over a period oftime, in addition to aliquots of the suspension removed randomly andanalyzed for protein concentration. In addition, the images taken byoptical microscopy can show the dominant forces on the particles atlower concentrations and whether the particles are aggregating,flocculating or repulsing. Particle size measurements over time willshow the effects of Oswald ripening and coagulation of particles. Tocomplete the study, the electrophoretic mobility of the particles willbe measured and quantify the zeta potential, which will determine theelectrostatic stabilization of the particles in the suspension.

The final step is to determine the stability of Mab in the formulatedsuspension. For aqueous based suspensions, appropriate dilutions must berun to produce a solution of the Mab at the necessary concentration foranalysis. The Mab in solution will also be separated from the suspendedparticles and both will be analyzed separately for stability. Fornon-aqueous based suspensions an aliquot with sufficient Mabconcentration must be exposed to pure water or a suitable buffer toallow the suspended particles to partition to the aqueous phase insolution without denaturing on the oil-water interface. As has been donepreviously, suspensions will be exposed to the appropriate aqueousbuffer for 1-3 days to allow slow partitioning and minimal exposure tothe potentially denaturing oil-water interface.1 In addition, standardswill be run to ensure that the measurements are an accuraterepresentation of the suspended particles.1 The stability of the Mab canthen be characterized by a variety of techniques includingsize-exclusion chromatography (SEC), dynamic light scattering (DLS),analytical ultracentrifugation (AUC), and optical turbidity for solubleand insoluble aggregates.

Moisture content of both the dried powder and the reconstitutednon-aqueous suspension will be analyzed over time to determine theeffect of excess water on the stability of the formulation. As has beennoted in previous experiments, at low levels of protein hydration, thewater sorption over time in a non-polar or moderately polar organicsolvent is similar to that from the vapor phase itself. 12 In addition,a Mab specific ELISA assay can be run to demonstrate the % activity ofthe Mab after formulation to analyze misfolding and denaturation of theMab. This technique has been used previously to observe the resultingbinding affinity of an IgG after reconstitution. FTIR can also be run todetermine any Mab-excipient interactions with any part of the finalformulation.

Production of particles by film freezing in vials. In many particleformation processes, the transfer of solids from various surfaces tovials for delivery of the final dosage form presents problems. It isnecessary to maintain sterile conditions and it can be difficult todetermine and control the exact amount of particles transferred. Inaddition, the particle size may change during handling. It would bedesirable to produce the particles directly within the vial the finaldosage form will be stored in. This process of direct freezing andparticle production within a vial is practiced in conventional traylyophilization. However, the slow freezing rates by heat transferthrough the shelf to the vial leads to particles typically on the orderof hundreds of microns. The current technique provides a means toproduce and control sub-micron and micron sized particle distributions.

A method is provided for forming particles of substances inside vials,the method comprising: (a) introducing a liquid solution of thesubstances inside a cylindrical vial; (b) immersing the cylindrical vialinto a liquid coolant while rotating it horizontally until the liquidhas frozen as a film in the vial's internal walls; (c) removal of thesolvent by lyophilization or by extraction of the frozen solvent into asecond solvent.

Thus, the current invention provides an alternative to freeze much morequickly with a submersion of the vials in a cryogenic liquid than in thecase of tray lyophilization. The more rapid freezing on the order of 20s can result in sub-micron particles. The thickness of the freezingliquid, in the range of 0.2 to 4 mm, facilitates the rapid freezing. Thevials may be transferred directly to a lyophilizer to remove the solventwhile producing the final particles in the vial. The final particles canalso be produced by addition of agents (salts, a second solvent,polymers, etc.) to decrease the solubility of the particles to produce asuspension of the particles. Thus the solids never have to be removedfrom the vial.

This method permits the production of particles with fine control of thesize distribution by freezing liquid solutions of substances in thinuniform films at fast freezing rates inside vials. The method isimplemented in three fundamental steps: (a) introducing a liquidsolution of the substances inside a cylindrical vial; (b) immersing thecylindrical vial into a liquid coolant while rotating it horizontallyuntil the liquid has frozen as a film in the vial's internal walls; (c)removal of the solvent by lyophilization or by extraction of the frozensolvent into a second solvent.

The first step begins by dissolving the active substances in an aqueoussolution in typical concentrations ranging from 1 mg/ml to 500 mg/ml.This solution may also contain excipients including cryoprotectives orsurfactants as an example. The solution is introduced into a cylindricalvial, where the liquid volume and the vial's dimensions determine thethickness of the final frozen film (important variables in the controlof the size distribution). Table 14 shows an example of different filmthicknesses obtained for vials of two different sizes. Table 14. Filmthicknesses obtained after freezing different volumes of liquid solutionin vials of different dimensions.

Vial 1 Vial 2 Internal Diameter 15 mm Internal Diameter 24 mm Length 40mm Length 48 mm Liquid Volume Film thickness Liquid Volume Filmthickness (ml) (mm) (ml) (mm) 1 0.6 1 0.3 2 1.2 2 0.6 3 1.8 3 0.9 5 3.55 1.5 10 3.2

The second step includes immersing the vial horizontally inside liquidcoolant (e.g. liquid N2) while rotating it. The rotation causes theliquid solution to freeze as a film of uniform thickness in thecylindrical vial internal walls. The coolant temperature (typicallyranging from 50 K to 253 K) and the rotation speed (typically rangingfrom 15 RPM to 600 RPM) may be adjusted to control the freezing rate.FIG. 18 show freezing temperature profiles measured for freezingdifferent liquid volumes inside vials. The temperatures were measuredwith a type T thermocouple while processing with a coolant at 80 K and arotation speed of 30 RPM.

The third step is the removal of the solvent by lyophilization or addingagents to the frozen solvent to create a poorly-soluble environmentproducing a suspension. A second solvent, salts, polymers and otheragents can be added to the aqueous based formulation to produce apoorly-soluble environment for the protein-based particles. Solvents aretypically water-miscible organic solvents such as acetonitrile andethanol. Salts, such as sodium sulphate and ammonium sulphate, andpolymers such as PEG, cause a decrease in the solubility of the proteinsin an aqueous environment, creating a suspension of particles. FIG. 19shows typical particle size distributions obtained with the presentmethod measured by a multiangle laser light scattering with a MalvernMastersizer-S with the particles suspended in acetonitrile. At selectedconditions, nanometric particles, micrometric or bimodal distributionsof particles of both size scales can be produced, as shown in FIG. 19.The size distribution is controlled by solutes concentration, thetemperature of the liquid coolant, and the volume of the liquid and thevial rotating speed. Table 15 shows the process conditions that resultedin the particle size distributions shown in FIG. 19.

TABLE 15 Process conditions used in the production of particles by FilmFreezing into Vials, corresponding to the size distributions shown inFIG. 13. The film thickness is defined as the maximum thickness of thefinal frozen mass within a vial in the horizontal position. The vialinside diameter is 15 mm. Protein Coolant REF. in Concentration FilmThickness Rotation speed Temperature FIG. 7 mg/ml mm RPM K (a) 20 2.6 3080 (b) 10 0.6 30 80 (c) 5 2.6 120 80 (d) 5 0.6 30 210

The following examples demonstrate protein suspensions made by filmfreezing of protein in a vial followed by lyophilization, and thensuspension of the lyophilized material in a solvent with manual shaking.In FIG. 20, 4 ml of lysozyme solution (20 mg/ml) in water were frozen byFilm Freezing inside the Vial. After lyophilization, a suspension with80 mg/mL lysozyme was formed by adding benzyl benzoate. In FIG. 21, 2 mlof hemoglobin solution (150 mg/ml) in water was frozen by Film Freezinginside the Vial. The frozen solution was lyophilized and the particleswere suspended in 2 ml of Benzyl Benzoate to make up a 150 mg/mlsuspension. In both cases, the suspensions did not settle over 1 day,and could resuspended by manual shaking.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1.-50. (canceled)
 51. A method of delivering a suspension of proteinparticles to a subject, the method comprising subcutaneously injectingsaid suspension into said subject, wherein the protein concentrationwithin said suspension is greater than 200 mg/mL and the viscosity ofsaid suspension is less than 50 centipoise.
 52. The method of claim 51,wherein said injection is accomplished using a syringe comprising a21-gauge to 27-gauge needle.
 53. The method of claim 52, wherein saidsyringe comprises a 25-gauge to 27-gauge needle.
 54. The method of claim51, wherein said suspension has a volume of 5 mL or less.
 55. The methodof claim 51, wherein said suspension has a volume of 1.5 mL or less. 56.The method of claim 51, wherein said particles are sub-micron sizedparticles.